Implantable matrix having optimum ligand concentrations

ABSTRACT

Implantable matrices and methods are provided. The matrices are configured to fit at or near a target tissue site, the matrices comprise biodegradable materials and ligands bound to the matrices and are configured to bind receptors and allow influx of cells into the implantable matrices, wherein the ratio of ligands to receptors is from about 1.5 to about 0.5.

BACKGROUND

Implantable matrices have been used extensively to solve various medicalproblems in human and animal orthopedic surgical practices and theirapplication has also extended to the field of cosmetic andreconstructive surgery, dental reconstructive surgery, and other medicalfields involving surgery of hard and soft tissues.

Often times, to enhance growth of different cells within the implantablematrix and repair of tissue, a ligand is disposed on the implantablematrix and becomes available so that it can interact with a particulartarget receptor and cause the desired biological function. The formationand dissociation of specific noncovalent interactions between a ligandand its receptor plays a crucial role in the function of biologicalsystems. The ligand and/or receptor of the matrix, in some embodiments,can stimulate other mammalian cell growth, such as for example,myocytes, cardiocytes to repair heart tissue, neuronal cells, or thelike.

For example, when dealing with a ligand, such as bone morphogenicprotein (BMP), it can be disposed on an implantable matrix and placed ina bone defect. BMP can be applied to the matrix before, during or afterimplantation. The BMP interacts with specific receptors on the cellsurface, referred to as bone morphogenetic protein receptors (BMPRs). Aspersons of ordinary skill are aware the BMP spur the patient's body tobegin the formation of new bone and/or cartilage growth. The BMP actsmuch like a catalyst, encouraging the necessary cells (including, butnot limited to, mesenchymal stem cells, osteoblasts, and osteoclasts) tomore rapidly migrate into the matrix, which is eventually resorbed via acell-mediated process and newly formed bone is deposited at or near thebone defect. In this manner severe fractures may be healed, andvertebrae successfully fused.

Signal transduction through BMPRs also results in mobilization ofmembers of the SMAD family of proteins. The signaling pathways involvingBMPs, BMPRs and SMADs are also important in the development of theheart, and central nervous system tissue, as well as bone and cartilage.

Sometimes when too much ligand is applied to the matrix, or the surgeonmanipulates the matrix to place it in the defect, excessive compressionoccurs causing increased amounts of ligand (e.g., bone morphogenicprotein, TGF-alpha, TGF-beta, EGF, etc.) to leak from the matrix, whichmay reduce the stable microenvironment for cell growth, cause off targetside effects (e.g., unwanted cell growth in other areas) or inhibitbiological activity via a feedback inhibition. Sometimes too littleligand is applied to the matrix or the ligand on or in the matrix isdepleted, which may also reduce the stable microenvironment for cellgrowth. Thus, there is a need to develop new matrices that have optimumligand receptor concentrations.

SUMMARY

Implantable matrices are provided that improve efficacy and maintain astable microenvironment for cell growth. By maintaining a ligand toreceptor ratio of from about 0.5 to about 1.5 a stable microenvironmentfor cell growth can be achieved. In some embodiments, the implantablematrices provided minimize off target side effects and reduce unwantedfeed back inhibition.

In some embodiments, the matrix comprises bone morphogenic protein, andmaintains a ligand to receptor ratio of from about 0.5 to about 1.5,which will reduce excess ligand from being forced out of the matrix intothe surrounding environment, which may lead to unwanted adverse eventssuch as local transient bone resorption.

In some embodiments, there is an implantable matrix configured to fit ator near a target tissue site, the matrix comprising: a biodegradablematerial and a ligand bound to the matrix and configured to bind areceptor and allow influx of cells into the implantable matrix, whereinthe ratio of ligand to receptor is from about 0.5 to about 1.5.

In some embodiments, there is a method for treating a target tissue sitebeneath the skin in a patient in need of such treatment, the methodcomprising administering an implantable matrix configured to fit at ornear a target tissue site, the matrix comprising: a biodegradablecollagen and a ligand comprising bone morphogenic protein bound to thematrix and configured to bind a receptor of progenitor, bone and/orcartilage cells and allow influx of the cells into the implantablematrix, wherein the ratio of ligand to receptor is from about 0.5 toabout 1.5.

In some embodiments, there is a method of making an implantable matrixconfigured to fit at or near a target tissue site, the method comprisingproviding a biodegradable material and applying a ligand to bind theligand to the matrix, the matrix configured to bind a receptor and allowinflux of cells into the implantable matrix, wherein the ligand isapplied to the matrix in an amount where the ratio of ligand to receptoris from about 0.5 to about 1.5.

Additional features and advantages of various embodiments will be setforth in part in the description that follows, and in part will beapparent from the description, or may be learned by practice of variousembodiments. The objectives and other advantages of various embodimentswill be realized and attained by means of the elements and combinationsparticularly pointed out in the description and appended claims.

BRIEF DESCRIPTION OF THE FIGURES

In part, other aspects, features, benefits and advantages of theembodiments will be apparent with regard to the following description,appended claims and accompanying drawings where:

FIG. 1 illustrates an axial cross-sectional view of the implantablematrix being injected at a target tissue site and the optimum ligandconcentration being placed in the matrix.

FIG. 2 illustrates a longitudinal cross-sectional view of soft tissueand sub-chondral bone where the matrix is implanted into a bone defect.The matrix contains a ligand (e.g., growth factor) that interacts withthe receptors of the bone.

It is to be understood that the figures are not drawn to scale. Further,the relation between objects in a figure may not be to scale, and may infact have a reverse relationship as to size. The figures are intended tobring understanding and clarity to the structure of each object shown,and thus, some features may be exaggerated in order to illustrate aspecific feature of a structure.

DETAILED DESCRIPTION

For the purposes of this specification and appended claims, unlessotherwise indicated, all numbers expressing quantities of ingredients,percentages or proportions of materials, reaction conditions, and othernumerical values used in the specification and claims, are to beunderstood as being modified in all instances by the term “about.”Accordingly, unless indicated to the contrary, the numerical parametersset forth in the following specification and attached claims areapproximations that may vary depending upon the desired propertiessought to be obtained by the present application. At the very least, andnot as an attempt to limit the application of the doctrine ofequivalents to the scope of the claims, each numerical parameter shouldat least be construed in light of the number of reported significantdigits and by applying ordinary rounding techniques.

Notwithstanding that the numerical ranges and parameters setting forththe broad scope of the invention are approximations; the numericalvalues are as precise as possible. Any numerical value, however,inherently contains certain errors necessarily resulting from thestandard deviation found in their respective testing measurements.Moreover, all ranges disclosed herein are to be understood to encompassany and all subranges subsumed therein. For example, a range of “1 to10” includes any and all subranges between (and including) the minimumvalue of 1 and the maximum value of 10, that is, any and all subrangeshaving a minimum value of equal to or greater than 1 and a maximum valueof equal to or less than 10, e.g., 5.5 to 10.

Additionally, unless defined otherwise or apparent from context, alltechnical and scientific terms used herein have the same meanings ascommonly understood by one of ordinary skill in the art to which thisinvention belongs.

Unless explicitly stated or apparent from context, the following termsor phrases have the definitions provided below:

DEFINITIONS

It is noted that, as used in this specification and the appended claims,the singular forms “a,” “an,” and “the,” include plural referents unlessexpressly and unequivocally limited to one referent. Thus, for example,reference to “a matrix” includes one, two, three or more matrices.

The term “biodegradable” includes that all or parts of the matrix willdegrade over time by the action of enzymes, by hydrolytic action and/orby other similar mechanisms in the human body. In various embodiments,“biodegradable” includes that a matrix (e.g., sponge, sheet, etc.) canbreak down or degrade within the body to non-toxic components after orwhile a therapeutic agent has been or is being released. By“bioerodible” it is meant that the matrix will erode or degrade overtime due, at least in part, to contact with substances found in thesurrounding tissue, fluids or by cellular action. By “bioabsorbable” or“bioresorbable” it is meant that the matrix will be broken down andabsorbed within the human body, for example, by a cell or tissue.“Biocompatible” means that the matrix will not cause substantial tissueirritation or necrosis at the target tissue site.

The term “mammal” refers to organisms from the taxonomy class“mammalian,” including but not limited to humans, other primates such aschimpanzees, apes, orangutans and monkeys, rats, mice, cats, dogs, cows,horses, etc.

The term “resorbable” includes biologic elimination of the products ofdegradation by metabolism and/or excretion over time, for example,usually months.

The term “particle” refers to pieces of a substance of all shapes,sizes, thickness and configuration such as fibers, threads, narrowstrips, thin sheets, clips, shards, etc., that posses regular, irregularor random geometries. In some embodiments, the particles are elongatedhaving more length than width (e.g., long and slender particles). Itshould be understood that some variation in dimension will occur in theproduction of the particles and particles demonstrating such variabilityin dimensions are within the scope of the present application.

The term “target tissue site” is intended to mean the location of thetissue to be treated. Typically the placement site of the matrix will bethe same as the target site to provide for optimal targeted drugdelivery. However, the present application also contemplates positioningthe matrix at a placement site at or near the target site such that theligand (e.g., growth factor, other therapeutic agent) can be deliveredto the surrounding vasculature, which carries the agent to the desirednearby target site. As used herein, the term “at or near” includesembodiments where the placement site and target site are within closeproximity (e.g., within about 1 mm to 5 cm).

The term “autograft” as utilized herein refers to tissue that isextracted from the intended recipient of the implant. These includeelongated particles.

The term “allograft” as utilized herein refers to tissue intended forimplantation that is taken from a different member of the same speciesas the intended recipient. These include elongated particles.

The term “xenogenic” as utilized herein refers to material intended forimplantation obtained from a donor source of a different species thanthe intended recipient. For example, when the implant is intended foruse in an animal such as a horse (equine), xenogenic tissue of, e.g.,bovine, porcine, caprine, etc., origin may be suitable. These includeelongated particles.

The term “transgenic” as utilized herein refers to tissue intended forimplantation that is obtained from an organism that has been geneticallymodified to contain within its genome certain genetic sequences obtainedfrom the genome of a different species. The different species is usuallythe same species as the intended implant recipient but such limitationis merely included by way of example and is not intended to limit thedisclosure here in anyway whatsoever. These include elongated particles.

The expressions “whole bone” and “substantially fully mineralized bone”refer to bone containing its full or substantially full, originalmineral content that can be used. This type of bone can be used to makeelongated particles.

The expression “demineralized bone” includes bone that has beenpartially, fully, segmentally or superficially (surface) demineralized.This type of bone can be used to make elongated particles.

The expression “substantially fully demineralized bone” as utilizedherein refers to bone containing less than about 8% of its originalmineral context. This type of bone can be used to make elongatedparticles.

A “therapeutically effective amount” or “effective amount” is such thatwhen administered, the ligand (e.g., drug, growth factor, etc.) resultsin alteration of the biological activity, such as, for example,promotion of bone, cartilage and/or other tissue (e.g., vascular tissue)growth, inhibition of inflammation, reduction or alleviation of pain,improvement in the condition through inhibition of an immunologicresponse, etc. The dosage administered to a patient can be as single ormultiple doses depending upon a variety of factors, including theligand's administered pharmacokinetic properties, the route ofadministration, patient conditions and characteristics (sex, age, bodyweight, health, size, etc.), extent of symptoms, concurrent treatments,frequency of treatment and the effect desired. In some embodiments theimplantable matrix is designed for sustained release. In someembodiments, the implantable matrix comprises an effective amount of aligand that is based on a ratio of ligand to receptor. In someembodiments, the ratio of ligand to receptor is from about 0.5 to about1.5.

The phrase “immediate release” is used herein to refer to one or moreligand(s) (e.g., therapeutic agent(s)) that is introduced into the bodyand that is allowed to dissolve in or become absorbed at the location towhich it is administered, with no intention of delaying or prolongingthe dissolution or absorption of the drug.

The phrases “prolonged release”, “sustained release” or “sustainrelease” (also referred to as extended release or controlled release)are used herein to refer to one or more ligand(s) (e.g., therapeuticagent(s)) that is introduced into the body of a human or other mammaland continuously or continually releases a stream of one or moretherapeutic agents over a predetermined time period and at a therapeuticlevel sufficient to achieve a desired therapeutic effect throughout thepredetermined time period. Reference to a continuous or continualrelease stream is intended to encompass release that occurs as theresult of biodegradation in vivo of the matrix and/or component thereof,or as the result of metabolic transformation or dissolution of thetherapeutic agent(s) or conjugates of therapeutic agent(s). The releaseneed not be linear and can be pulse type dosing.

The “matrix” of the present application is utilized as a scaffold forbone and/or cartilage repair, regeneration, and/or augmentation.Typically, the matrix provides a 3-D matrix of interconnecting pores,which acts as a pliant scaffold for cell migration. The morphology ofthe matrix guides cell migration and cells are able to migrate into orover the matrix, respectively. The cells then are able to proliferateand synthesize new tissue and form bone and/or cartilage. In someembodiments, the matrix is resorbable.

In some embodiments, the matrix can be shaped. The term “shaped”includes that the matrix including the elongated particles is formedinto sheets, plates, disks, cones, pins, screws, tubes, teeth, bones,portion of bone, wedges, cylinders, threaded cylinders, and the like, aswell as more complex geometric configurations.

The terms “treating” and “treatment” when used in connection with adisease or condition refer to executing a protocol that may include arepair procedure (e.g., osteochondral repair procedure), administeringone or more matrices to a patient (human or other mammal), in an effortto alleviate signs or symptoms of the disease or condition orimmunological response. Alleviation can occur prior to signs or symptomsof the disease or condition appearing, as well as after theirappearance. Thus, treating or treatment includes preventing orprevention of disease or undesirable condition. In addition, treating,treatment, preventing or prevention do not require complete alleviationof signs or symptoms, does not require a cure, and specifically includesprotocols that have only a marginal effect on the patient. In someembodiments, the implantable matrix can be used to treat subchondral,osteochondral, hyaline cartilage and/or condyle defects.

The term “subchondral” includes an area underlying joint cartilage. Theterm “subchondral bone” includes a very dense, but thin layer of bonejust below a zone of cartilage and above the cancellous or trabecularbone that forms the bulk of the bone structure of the limb.“Osteochondral” includes a combined area of cartilage and bone where alesion or lesions can occur. “Osteochondral defect” includes a lesion,which is a composite lesion of cartilage and subchondral bone. “Hyalinecartilage” includes cartilage containing groups of isogenouschondrocytes located within lacunae cavities which are scatteredthroughout an extracellular collagen matrix. A “condyle” includes arounded articular surface of the extremity of a bone.

The matrix may be osteogenic. The term “osteogenic” as used hereinincludes the ability of the matrix to enhance or accelerate the growthof new bone tissue by one or more mechanisms such as osteogenesis,osteoconduction and or osteoinduction. In some embodiments, the matrixis osteogenic and can be delivered to other surgical sites, particularlysites at which bone growth is desired. These include, for instance, therepair of spine (e.g., vertebrae fusion) cranial defects, iliac crestback-filling, acetabular defects, in the repair of tibial plateau, longbone defects, spinal site defects or the like. Such methods can be usedto treat major or minor defects in these or other bones caused by trauma(including open and closed fractures), disease, or congenital defects,for example.

The matrix may be osteoinductive. The term “osteoinductive” as usedherein includes the ability of a substance to recruit cells from thehost that have the potential for forming new bone and repairing bonetissue. Most osteoinductive materials can stimulate the formation ofectopic bone in soft tissue.

The matrix may be osteoconductive. The term “osteoconductive” asutilized herein includes the ability of a non-osteoinductive substanceto serve as a suitable template or substrate along which bone may grow.

The matrix may be implantable. The term “implantable” as utilized hereinrefers to a biocompatible device retaining potential for successfulplacement within a mammal. The expression “implantable device” andexpressions of like import as utilized herein refers to any objectimplantable through surgery, injection, or other suitable means whoseprimary function is achieved either through its physical presence ormechanical properties.

The term “carrier” includes a diluent, adjuvant, buffer, excipient, orvehicle with which a composition can be administered. Carriers caninclude sterile liquids, such as, for example, water and oils, includingoils of petroleum, animal, vegetable or synthetic origin, such as, forexample, peanut oil, soybean oil, mineral oil, sesame oil, or the like.The growth factor may include a carrier.

The term “excipient” includes a non-therapeutic agent added to apharmaceutical composition to provide a desired consistency orstabilizing effect. Excipients for parenteral formulations, include, forexample, oils (e.g., canola, cottonseed, peanut, safflower, sesame,soybean), fatty acids and salts and esters thereof (e.g., oleic acid,stearic acid, palmitic acid), alcohols (e.g., ethanol, benzyl alcohol),polyalcohols (e.g., glycerol, propylene glycols and polyethyleneglycols, e.g., PEG 3350), polysorbates (e.g., polysorbate 20,polysorbate 80), gelatin, albumin (e.g., human serum albumin), salts(e.g., sodium chloride), succinic acid and salts thereof (e.g., sodiumsuccinate), amino acids and salts thereof (e.g., alanine, histidine,glycine, arginine, lysine), acetic acid or a salt or ester thereof(e.g., sodium acetate, ammonium acetate), citric acid and salts thereof(e.g., sodium citrate), benzoic acid and salts thereof, phosphoric acidand salts thereof (e.g., monobasic sodium phosphate, dibasic sodiumphosphate), lactic acid and salts thereof, polylactic acid, glutamicacid and salts thereof (e.g., sodium glutamate), calcium and saltsthereof (e.g., CaCl₂, calcium acetate), phenol, sugars (e.g., glucose,sucrose, lactose, maltose, trehalose), erythritol, arabitol, isomalt,lactitol, maltitol, mannitol, sorbitol, xylitol, nonionic surfactants(e.g., TWEEN 20, TWEEN 80), ionic surfactants (e.g., sodium dodecylsulfate), chlorobutanol, DMSO, sodium hydroxide, glycerin, m-cresol,imidazole, protamine, zinc and salts thereof (e.g, zinc sulfate),thimerosal, methylparaben, propylparaben, carboxymethylcellulose,chlorobutanol, or heparin. The growth factor may include an excipient.

The term “lyophilized” or “freeze-dried” includes a state of a substancethat has been subjected to a drying procedure such as lyophilization,where at least 50% of moisture has been removed. The matrix and/orligand may be lyophilized or freeze-dried.

A “preservative” includes a bacteriostatic, bacteriocidal, fungistaticor fungicidal compound that is generally added to formulations to retardor eliminate growth of bacteria or other contaminating microorganisms inthe formulations. Preservatives include, for example, benzyl alcohol,phenol, benzalkonium chloride, m-cresol, thimerosol, chlorobutanol,methylparaben, propylparaben and the like. Other examples ofpharmaceutically acceptable preservatives can be found in the USP. Theligand and/or matrix may have preservatives or be preservative free.

As used herein, the term “ligand” refers to a molecule that canselectively bind to a receptor. The term selectively means that thebinding interaction is a specific interaction as opposed to anon-specific interaction. A ligand can be a peptide, polypeptide,nucleic acid, carbohydrate, lipid, or any organic derived compound.Moreover, derivatives, analogues and mimetic compounds are also intendedto be included within the definition of this term. As such, a moleculethat is a ligand can also be a receptor and, conversely, a molecule thatis a receptor can also be a ligand since ligands and receptors aredefined as binding partners. Specific examples of ligands are natural orsynthetic organic compounds as well as recombinantly or syntheticallyproduced polypeptides. For example, ligands include, but are not limitedto, BMP (bone morphogenetic protein), BMP-2 (bone morphogeneticprotein), bFGF (basic fibroblast growth factor), IGF-1 (insulin-likegrowth factor), PDGF (platelet-derived growth factor), rhBMP (humanrecombinant bone morphogenetic protein), TGF-β 1 (transforming growthfactor beta 1), VEGF (vascular endothelial growth factor), GDf (growthand differentiation factor), and any combinations thereof. The ligand isthe binding partner of the receptor.

As used herein, the term “polypeptide” when used in reference to areceptor or a ligand is intended to refer to peptide, polypeptide orprotein of two or more amino acids. The term is similarly intended torefer to derivatives, analogues and functional mimetics thereof. Forexample, derivatives can include chemical modifications of thepolypeptide such as alkylation, acylation, carbamylation, iodination, orany modification which derivatizes the polypeptide. Analogues caninclude modified amino acids, for example, hydroxyproline orcarboxyglutamate, and can include amino acids that are not linked bypeptide bonds. Mimetics encompass chemicals containing chemical moietiesthat mimic the function of the polypeptide regardless of the predictedthree-dimensional structure of the compound. For example, if apolypeptide contains two charged chemical moieties in a functionaldomain, a mimetic places two charged chemical moieties in a spatialorientation and constrained structure so that the charged chemicalfunction is maintained in three-dimensional space. Thus, all of thesemodifications are included within the term “polypeptide” so long as thepolypeptide retains its binding function.

As used herein, the term “receptor” is intended to refer to a moleculeof sufficient size so as to be capable of selectively binding a ligand.Such molecules generally are macromolecules, such as polypeptides,nucleic acids, carbohydrate or lipid. However, derivatives, analoguesand mimetic compounds as well as natural or synthetic organic compoundsare also intended to be included within the definition of this term. Thereceptor can be a fragment of the entire molecule so long as it exhibitsselective binding to a desired ligand. For example, if the receptor is apolypeptide, a fragment or domain of the native polypeptide whichmaintains substantially the same binding selectivity as the intactpolypeptide is intended to be included within the definition of the termreceptor. Specific examples of such a binding domain or fragment is thevariable region of an antibody molecule. Complementarity determiningregions (CDR) within the variable region can also exhibit substantiallythe same binding selectivity as the antibody molecule and are thereforeconsidered to be within the meaning of the term. The receptor is thebinding partner of the ligand.

Reference will now be made in detail to certain embodiments of theinvention. While the invention will be described in conjunction with theillustrated embodiments, it will be understood that they are notintended to limit the invention to those embodiments. On the contrary,the invention is intended to cover all alternatives, modifications, andequivalents that may be included within the invention as defined by theappended claims.

Implantable matrices are provided that improve efficacy and maintain astable microenvironment for cell growth. By maintaining a ligand toreceptor ratio of from about 0.5 to about 1.5 a stable microenvironmentfor cell growth can be achieved. In some embodiments, the implantablematrices provided minimize off target side effects and reduce unwantedfeed back inhibition.

In some embodiments, the matrix comprises bone morphogenic protein, andmaintains a ligand to receptor ratio of from about 0.5 to about 1.5,which will reduce excess ligand from being forced out of the matrix intothe surrounding environment, which may lead to unwanted adverse eventssuch as local transient bone resorption.

In some embodiments, the growth factor (e.g., rhBMP-2) will be moreevenly distributed throughout the interior of the matrix and facilitatemore uniform bone growth throughout the whole matrix. In someembodiments, the growth factor (e.g., rhBMP-2) is temporarily retainedwithin the matrix so as to limit new bone formation to within thematrix.

The headings below are not meant to limit the disclosure in any way;embodiments under any one heading may be used in conjunction withembodiments under any other heading.

Matrix

The matrix provides a tissue scaffold for the cells to guide the processof tissue formation in vivo in three dimensions. The morphology of thematrix guides cell migration and cells are able to migrate into or overthe matrix. The cells then are able to proliferate and synthesize newtissue and form bone and/or cartilage. In some embodiments, one or moretissue matrices are stacked on one another.

The matrix is porous and configured to allow influx of at least boneand/or cartilage cells therein. In some embodiments, the matrix is alsoconfigured to release a ligand. By porous is meant that the matrix has aplurality of pores. The pores of the matrix are a size large enough toallow influx of blood, other bodily fluid, and progenitor and/or boneand/or cartilage cells into the interior to guide the process of tissueformation in vivo in three dimensions.

In some embodiments, the matrix comprises a plurality of pores. In someembodiments, at least 10% of the pores are between about 50 micrometersand about 500 micrometers at their widest points. In some embodiments,at least 20% of the pores are between about 50 micrometers and about 250micrometers at their widest points. In some embodiments, at least 30% ofthe pores are between about 50 micrometers and about 150 micrometers attheir widest points. In some embodiments, at least 50% of the pores arebetween about 10 micrometers and about 500 micrometers at their widestpoints. In some embodiments, at least 90% of the pores are between about50 micrometers and about 250 micrometers at their widest points. In someembodiments, at least 95% of the pores are between about 50 micrometersand about 150 micrometers at their widest points. In some embodiments,100% of the pores are between about 10 micrometers and about 500micrometers at their widest points.

In some embodiments, the matrix has a porosity of at least about 30%, atleast about 50%, at least about 60%, at least about 70%, at least about90% or at least about 95%, or at least about 99%. The pores may supportingrowth of cells, formation or remodeling of bone, cartilage and/orvascular tissue. In some embodiments, the matrix contains one or moreligands bound in or on its surface.

In some embodiments, the porous interior can hold the ligand within thematrix and because the interior is porous, the ligand is evenlydistributed throughout the matrix when growth factor is injected intothe matrix.

In some embodiments, the ligand (e.g., growth factor) will be heldwithin the interior of the matrix and released into the environmentsurrounding the matrix (e.g., bone defect, osteochondral defect, etc.)as the matrix degrades over time

In some embodiments, the matrix comprises biodegradable polymeric andnon-polymeric material. For example, the matrix may comprises one ormore poly (alpha-hydroxy acids), poly (lactide-co-glycolide) (PLGA),polylactide (PLA), poly(L-lactide), polyglycolide (PG), polyethyleneglycol (PEG) conjugates of poly (alpha-hydroxy acids), polyorthoesters(POE), polyaspirins, polyphosphagenes, collagen, hydrolyzed collagen,gelatin, hydrolyzed gelatin, fractions of hydrolyzed gelatin, elastin,starch, pre-gelatinized starch, hyaluronic acid, chitosan, alginate,albumin, fibrin, vitamin E analogs, such as alpha tocopheryl acetate,d-alpha tocopheryl succinate, D,L-lactide, or L-lactide, -caprolactone,dextrans, vinylpyrrolidone, polyvinyl alcohol (PVA), PVA-g-PLGA,PEGT-PBT copolymer (polyactive), methacrylates, poly(N-isopropylacrylamide), PEO-PPO-PEO (pluronics), PEO-PPO-PAAcopolymers, PLGA-PEO-PLGA, PEG-PLG, PLA-PLGA, poloxamer 407,PEG-PLGA-PEG triblock copolymers, POE, SAIB (sucrose acetateisobutyrate), polydioxanone, methylmethacrylate (MMA), MMA andN-vinylpyyrolidone, polyamide, oxycellulose, copolymer of glycolic acidand trimethylene carbonate, polyesteramides, polyetheretherketone,polymethylmethacrylate, silicone, hyaluronic acid, chitosan, orcombinations thereof.

In some embodiments, the matrix can be designed to be compressionresistant and has elongated particles uniformly or randomly distributedthroughout it, and the matrix is substantially rigid and resistscompression. For example, in some embodiments, the matrix may have amodulus of elasticity between 1.0 MPa and 20.0 MPa, or 2.0 MPa and 10.0MPa or between 3.0 MPa and 5.0 MPa, with the higher MPa valuesobtainable by cross-linking.

In some embodiments, the matrix (e.g., exterior and/or interior)comprises collagen. Exemplary collagens include human or non-human(bovine, ovine, and/or porcine), as well as recombinant collagen orcombinations thereof. Examples of suitable collagen include, but are notlimited to, human collagen type I, human collagen type II, humancollagen type III, human collagen type IV, human collagen type V, humancollagen type VI, human collagen type VII, human collagen type VIII,human collagen type IX, human collagen type X, human collagen type XI,human collagen type XII, human collagen type XIII, human collagen typeXIV, human collagen type XV, human collagen type XVI, human collagentype XVII, human collagen type XVIII, human collagen type XIX, humancollagen type XXI, human collagen type XXII, human collagen type XXIII,human collagen type XXIV, human collagen type XXV, human collagen typeXXVI, human collagen type XXVII, and human collagen type XXVIII, orcombinations thereof. Collagen further may comprise hetero- andhomo-trimers of any of the above-recited collagen types. In someembodiments, the collagen comprises hetero- or homo-trimers of humancollagen type I, human collagen type II, human collagen type III, orcombinations thereof.

In some embodiments, the matrix comprises collagen-containingbiomaterials from the implant market which, when placed in a bonedefect, provide scaffolding around which the patient's new bone and/orcartilage will grow, gradually replacing the carrier matrix as thetarget site heals. Examples of suitable carrier matrices may include,but are not limited to, the MasterGraft® Matrix produced by MedtronicSofamor Danek, Inc., Memphis, Tenn.; MasterGraft® Putty produced byMedtronic Sofamor Danek, Inc., Memphis, Tenn.; Absorbable CollagenSponge (“ACS”) produced by Integra LifeSciences Corporation, Plainsboro,N.J.; bovine skin collagen fibers coated with hydroxyapatite, e.g.Healos®. marketed by Johnson & Johnson, USA; collagen sponges, e.g.Hemostagene® marketed by Coletica S A, France, or e.g. Helisat® marketedby Integra Life Sciences Inc., USA; and Collagraft® Bone Graft Matrixproduced by Zimmer Holdings, Inc., Warsaw, Ind.

Compression resistance is needed for many tissue engineeringapplications such as tibial plateau fractures, acetabular defects, longbone comminuted fractures, oral maxillofacial defects, spinal fusions,and cartilage subchondral defects. Compression resistant matrices willhelp facilitate adequate volumes of newly formed bone.

In some embodiments, the matrix is compression resistant where thematrix resists reduction in size or an increase in density when a forceis applied as compared to matrices without the elongated particlesdisposed in it. In various embodiments, the matrix resists compressionby at 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 95%, or more in one or all directions when a forceis applied to the matrix.

Ligands

Ligands include a substance that forms a complex with a biomolecule toserve a biological purpose. In some embodiments, a ligand can act as asignaling triggering biomolecule, binding to a site on a target protein.The binding may occur by intermolecular forces, such as ionic bonds,non-covalent bonds, hydrogen bonds and/or van der Waals forces. Thebinding is usually reversible. Ligand binding to a receptor alters thechemical conformation, that is the three dimensional shape of thereceptor. The conformational state of a receptor determines thefunctional state of a receptor.

In some embodiments, a ligand and/or therapeutic agent may be disposedon or in the matrix by hand, electrospraying, ionization spraying orimpregnating, lyophilization, vibratory dispersion (includingsonication), nozzle spraying, compressed-air-assisted spraying,injecting, brushing, soaking and/or pouring. For example, in someembodiments, when the ligand is a growth factor such as rhBMP-2, it maybe disposed on or in the biodegradable matrix by the surgeon before thebiodegradable matrix is administered or the matrix may be pre-loadedwith the growth factor by the manufacturer beforehand.

Ligands can be for example, small molecules, peptides, growth factors,cytokines, ligands, hormones, and other molecules that regulate growthand/or differentiation. The ligand can be captured from an autologoussource, be obtained from a commercial source, or can be manufactured(e.g., by recombinant procedures).

Examples of ligands that can be applied to or in the matrix include, butare not limited to, FGF, IGF, interleukins, IL-1, IL-11, TGF, NGF, EGF,HGF, simvastatsin, dexamethasone, oxysterols, sonic hedgehog,interferon, fibronectin, “RGD” or integrin peptides and/or protein,keratinocyte growth factor, osteogenic proteins, MSX1, NFκB1, RUNX2,SMAD1, SMAD2, SMAD3, SMAD4, SOX9, TNF, TWIST1, VDR., AHSG, AMBN, AMELY,BGLAP, ENAM, MINPP1, STATH, TUFT1, COL11A1, SOX9, ALPL, AMBN, AMELY,BGLAP, CALCR, CDH11, DMP1, DSPP, ENAM, MINPP1, PHEX, RUNX2, STATH,TFIP11, TUFT1, BGLAP, COL10A1, COL12A1, COL1A1, COL1A2, COL2A1, COMP,FGFR1, IGF1, IGF2, MSX1, ANXAS, CALCR, CDH11, COMP, DMP1, EGF, MMP2,MMP8, COL10A1, COL14A1, COL15A1, COL3A1, COL4A3, COL5A1, EGFR, FGF1,FGF3, IGF1R, CSF3, FLT1, IGF1, IGF1, IGF2, PDGFA, SMAD3, TGFB1, TGFB2,TGFB3, TGFBR2, CSF2, CSF3, FGFR1, FGFR2, FLT1, GDF10, IGF1, IGF1R, IGF2,PDGFA, TGFB1, TGFB2, TGFB3, TGFBR1, TGFBR2, VEGFA, VEGFB, AHSG,SERPINH1, CTSK, MMP10, MMP9, PHEX, AMBN, AMELY, ENAM, STATH, TUFT1, BGN,COMP, DSPP, GDF10, CDH11, ICAM1, ITGB1, VCAM1, ITGA1, ITGA2, ITGA3,ITGAM, ITGB1, CD36, COMP, SCARB1, AMH, GDF2 (BMP9), GDF3 (Vgr-2), GDFS(CDMP-1), GDF6, GDF7, IGFBP3, IL6, INHA (inhibin a), INHBA (inhibin BA),LEFTY1, LTBP1, LTBP2, LTBP4, NODAL, ACVR1 (ALK2), ACVR2A, ACVRL1 (ALK1),AMHR2, BMPR1A (ALK3), BMPR1B (ALK6), BMPR2, ITGBS (integrin B5), ITGB7(integrin B7), LTBP1, NROB1, STAT1, TGFB1I1, TGFBR1, (ALKS) TGFBR2,TGFBR3, TGFBRAP1, CDC25A, CDKN1A (p21WAF1/p21CIP1), CDKN2B (p15LNK2B),FOS, GSC (goosecoid), IGFBP3, ITGBS (integrin B5), ITGB7 (integrin B7),JUN, JUNB, MYC, SERPINE 1 (PAI-1), TGFB111, TSC22D1 (TGFB114), TGIF1,DLX2, ID1, ID2, JUNB, SOX4, STAT1, BAMBI, BMPER, CDKN2B (p15LNK2B), CER1(cerberus), CHRD (chordin), CST3, ENG (Evi-1), EVIL FKBP1B, HIPK2, NBL1(DAN), NOG, PLAU (uPA), RUNX1 (AML1), SMURF1 and other molecules thatregulate growth or differentiation, as well as combinations of theseligands.

The biodegradable matrix may comprise at least one ligand that is agrowth factor. These growth factors include osteoinductive agents (e.g.,agents that cause new bone growth in an area where there was none)and/or osteoconductive agents (e.g., agents that cause ingrowth of cellsinto and/or through the matrix). Osteoinductive agents can bepolypeptides or polynucleotides compositions. Polynucleotidecompositions of the osteoinductive agents include, but are not limitedto, isolated Bone Morphogenic Protein (BMP), Vascular Endothelial GrowthFactor (VEGF), Connective Tissue Growth Factor (CTGF), Osteoprotegerin,Growth Differentiation Factors (GDFs), Cartilage Derived MorphogenicProteins (CDMPs), Lim Mineralization Proteins (LMPs), Platelet derivedgrowth factor, (PDGF or rhPDGF), Insulin-like growth factor (IGF) orTransforming Growth Factor beta (TGF-beta) polynucleotides.Polynucleotide compositions of the osteoinductive agents include, butare not limited to, gene therapy vectors harboring polynucleotidesencoding the osteoinductive polypeptide of interest. Gene therapymethods often utilize a polynucleotide, which codes for theosteoinductive polypeptide operatively linked or associated to apromoter or any other genetic elements necessary for the expression ofthe osteoinductive polypeptide by the target tissue. Such gene therapyand delivery techniques are known in the art (see, for example,International Publication No. WO90/11092, the disclosure of which isherein incorporated by reference in its entirety). Suitable gene therapyvectors include, but are not limited to, gene therapy vectors that donot integrate into the host genome. Alternatively, suitable gene therapyvectors include, but are not limited to, gene therapy vectors thatintegrate into the host genome.

In some embodiments, the polynucleotide is delivered in plasmidformulations. Plasmid DNA or RNA formulations refer to polynucleotidesequences encoding osteoinductive polypeptides that are free from anydelivery vehicle that acts to assist, promote or facilitate entry intothe cell, including viral sequences, viral particles, liposomeformulations, lipofectin, precipitating agents or the like. Optionally,gene therapy compositions can be delivered in liposome formulations andlipofectin formulations, which can be prepared by methods well known tothose skilled in the art. General methods are described, for example, inU.S. Pat. Nos. 5,593,972, 5,589,466, and 5,580,859, the disclosures ofwhich are herein incorporated by reference in their entireties.

Gene therapy vectors further comprise suitable adenoviral vectorsincluding, but not limited to for example, those described in U.S. Pat.No. 5,652,224, which is herein incorporated by reference.

Polypeptide compositions of the isolated osteoinductive agents include,but are not limited to, isolated Bone Morphogenic Protein (BMP),Vascular Endothelial Growth Factor (VEGF), Connective Tissue GrowthFactor (CTGF), Osteoprotegerin, Growth Differentiation Factors (GDFs),Cartilage Derived Morphogenic Proteins (CDMPs), Lim MineralizationProteins (LMPs), Platelet derived growth factor, (PDGF or rhPDGF),Insulin-like growth factor (IGF) or Transforming Growth Factor beta(TGF-beta707) polypeptides. Polypeptide compositions of theosteoinductive agents include, but are not limited to, full lengthproteins, fragments or variants thereof.

Variants of the isolated osteoinductive agents include, but are notlimited to, polypeptide variants that are designed to increase theduration of activity of the osteoinductive agent in vivo. Typically,variant osteoinductive agents include, but are not limited to, fulllength proteins or fragments thereof that are conjugated to polyethyleneglycol (PEG) moieties to increase their half-life in vivo (also known aspegylation). Methods of pegylating polypeptides are well known in theart (See, e.g., U.S. Pat. No. 6,552,170 and European Pat. No. 0,401,384as examples of methods of generating pegylated polypeptides). In someembodiments, the isolated osteoinductive agent(s) are provided as fusionproteins. In one embodiment, the osteoinductive agent(s) are availableas fusion proteins with the Fc portion of human IgG. In anotherembodiment, the osteoinductive agent(s) are available as hetero- orhomodimers or multimers. Examples of some fusion proteins include, butare not limited to, ligand fusions between mature osteoinductivepolypeptides and the Fc portion of human Immunoglobulin G (IgG). Methodsof making fusion proteins and constructs encoding the same are wellknown in the art.

Isolated osteoinductive agents that are included within a matrix aretypically sterile. In a non-limiting method, sterility is readilyaccomplished for example by filtration through sterile filtrationmembranes (e.g., 0.2 micron membranes or filters). In one embodiment,the matrix includes osteoinductive agents comprising one or more membersof the family of Bone Morphogenic Proteins (“BMPs”). BMPs are a class ofproteins thought to have osteoinductive or growth-promoting activitieson endogenous bone tissue, or function as pro-collagen precursors. Knownmembers of the BMP family include, but are not limited to, BMP-1, BMP-2,BMP-3, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8, BMP-9, BMP-10, BMP-11, BMP-12,BMP-13, BMP-15, BMP-16, BMP-17, BMP-18 as well as polynucleotides orpolypeptides thereof, as well as mature polypeptides or polynucleotidesencoding the same.

BMPs utilized as osteoinductive agents comprise one or more of BMP-1;BMP-2; BMP-3; BMP-4; BMP-5; BMP-6; BMP-7; BMP-8; BMP-9; BMP-10; BMP-11;BMP-12; BMP-13; BMP-15; BMP-16; BMP-17; or BMP-18; as well as anycombination of one or more of these BMPs, including full length BMPs orfragments thereof, or combinations thereof, either as polypeptides orpolynucleotides encoding the polypeptide fragments of all of the recitedBMPs. The isolated BMP osteoinductive agents may be administered aspolynucleotides, polypeptides, full length protein or combinationsthereof.

In another embodiment, isolated osteoinductive agents that are loaded inthe matrix include osteoclastogenesis inhibitors to inhibit boneresorption of the bone tissue surrounding the site of implantation byosteoclasts. Osteoclast and osteoclastogenesis inhibitors include, butare not limited to, osteoprotegerin polynucleotides or polypeptides, aswell as mature osteoprotegerin proteins, polypeptides or polynucleotidesencoding the same. Osteoprotegerin is a member of the TNF-receptorsuperfamily and is an osteoblast-secreted decoy receptor that functionsas a negative regulator of bone resorption. This protein specificallybinds to its ligand, osteoprotegerin ligand (TNFSF11/OPGL), both ofwhich are key extracellular regulators of osteoclast development.

Osteoclastogenesis inhibitors that can be loaded in the matrix furtherinclude, but are not limited to, chemical compounds such asbisphosphonate, 5-lipoxygenase inhibitors such as those described inU.S. Pat. Nos. 5,534,524 and 6,455,541 (the contents of which are hereinincorporated by reference in their entireties), heterocyclic compoundssuch as those described in U.S. Pat. No. 5,658,935 (herein incorporatedby reference in its entirety), 2,4-dioxoimidazolidine and imidazolidinederivative compounds such as those described in U.S. Pat. Nos. 5,397,796and 5,554,594 (the contents of which are herein incorporated byreference in their entireties), sulfonamide derivatives such as thosedescribed in U.S. Pat. No. 6,313,119 (herein incorporated by referencein its entirety), or acylguanidine compounds such as those described inU.S. Pat. No. 6,492,356 (herein incorporated by reference in itsentirety).

In another embodiment, isolated osteoinductive agents that can be loadedin the matrix include one or more members of the family of ConnectiveTissue Growth Factors (“CTGFs”). CTGFs are a class of proteins thoughtto have growth-promoting activities on connective tissues. Known membersof the CTGF family include, but are not limited to, CTGF-1, CTGF-2,CTGF-4 polynucleotides or polypeptides thereof, as well as matureproteins, polypeptides or polynucleotides encoding the same.

In another embodiment, isolated osteoinductive agents that can be loadedin the matrix include one or more members of the family of VascularEndothelial Growth Factors (“VEGFs”). VEGFs are a class of proteinsthought to have growth-promoting activities on vascular tissues. Knownmembers of the VEGF family include, but are not limited to, VEGF-A,VEGF-B, VEGF-C, VEGF-D, VEGF-E or polynucleotides or polypeptidesthereof, as well as mature VEGF-A, proteins, polypeptides orpolynucleotides encoding the same.

In another embodiment, isolated osteoinductive agents that can be loadedin the matrix include one or more members of the family of TransformingGrowth Factor-beta (“TGFbetas”). TGF-betas are a class of proteinsthought to have growth-promoting activities on a range of tissues,including connective tissues. Known members of the TGF-beta familyinclude, but are not limited to, TGF-beta-1, TGF-beta-2, TGF-beta-3,polynucleotides or polypeptides thereof, as well as mature protein,polypeptides or polynucleotides encoding the same.

In another embodiment, isolated osteoinductive agents that can be loadedin the matrix include one or more Growth Differentiation Factors(“GDFs”). Known GDFs include, but are not limited to, GDF-1, GDF-2,GDF-3, GDF-7, GDF-10, GDF-11, and GDF-15. For example, GDFs useful asisolated osteoinductive agents include, but are not limited to, thefollowing GDFs: GDF-1 polynucleotides or polypeptides corresponding toGenBank Accession Numbers M62302, AAA58501, and AAB94786, as well asmature GDF-1 polypeptides or polynucleotides encoding the same. GDF-2polynucleotides or polypeptides corresponding to GenBank AccessionNumbers BC069643, BC074921, Q9UK05, AAH69643, or AAH74921, as well asmature GDF-2 polypeptides or polynucleotides encoding the same. GDF-3polynucleotides or polypeptides corresponding to GenBank AccessionNumbers AF263538, BC030959, AAF91389, AAQ89234, or Q9NR23, as well asmature GDF-3 polypeptides or polynucleotides encoding the same. GDF-7polynucleotides or polypeptides corresponding to GenBank AccessionNumbers AB158468, AF522369, AAP97720, or Q7Z4P5, as well as mature GDF-7polypeptides or polynucleotides encoding the same. GDF-10polynucleotides or polypeptides corresponding to GenBank AccessionNumbers BC028237 or AAH28237, as well as mature GDF-10 polypeptides orpolynucleotides encoding the same.

GDF-11 polynucleotides or polypeptides corresponding to GenBankAccession Numbers AF100907, NP_(—)005802 or 095390, as well as matureGDF-11 polypeptides or polynucleotides encoding the same. GDF-15polynucleotides or polypeptides corresponding to GenBank AccessionNumbers BC008962, BC000529, AAH00529, or NP_(—)004855, as well as matureGDF-15 polypeptides or polynucleotides encoding the same.

In another embodiment, isolated osteoinductive agents that can be loadedin the matrix include Cartilage Derived Morphogenic Protein (CDMP) andLim Mineralization Protein (LMP) polynucleotides or polypeptides. KnownCDMPs and LMPs include, but are not limited to, CDMP-1, CDMP-2, LMP-1,LMP-2, or LMP-3.

CDMPs and LMPs useful as isolated osteoinductive agents that can beloaded in the matrix include, but are not limited to, the followingCDMPs and LMPs: CDMP-1 polynucleotides and polypeptides corresponding toGenBank Accession Numbers NM_(—)000557, U13660, NP_(—)000548 or P43026,as well as mature CDMP-1 polypeptides or polynucleotides encoding thesame. CDMP-2 polypeptides corresponding to GenBank Accession Numbers orP55106, as well as mature CDMP-2 polypeptides. LMP-1 polynucleotides orpolypeptides corresponding to GenBank Accession Numbers AF345904 orAAK30567, as well as mature LMP-1 polypeptides or polynucleotidesencoding the same. LMP-2 polynucleotides or polypeptides correspondingto GenBank Accession Numbers AF345905 or AAK30568, as well as matureLMP-2 polypeptides or polynucleotides encoding the same. LMP-3polynucleotides or polypeptides corresponding to GenBank AccessionNumbers AF345906 or AAK30569, as well as mature LMP-3 polypeptides orpolynucleotides encoding the same.

In another embodiment, isolated osteoinductive agents that can be loadedin the matrix include one or more members of any one of the families ofBone Morphogenic Proteins (BMPs), Connective Tissue Growth Factors(CTGFs), Vascular Endothelial Growth Factors (VEGFs), Osteoprotegerin orany of the other osteoclastogenesis inhibitors, Growth DifferentiationFactors (GDFs), Cartilage Derived Morphogenic Proteins (CDMPs), LimMineralization Proteins (LMPs), or Transforming Growth Factor-betas(TGF-betas), as well as mixtures or combinations thereof.

In another embodiment, the one or more isolated osteoinductive agentsthat can be loaded in the matrix are selected from the group consistingof BMP-1, BMP-2, BMP-3, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8, BMP-9,BMP-10, BMP-11, BMP-12, BMP-13, BMP-15, BMP-16, BMP-17, BMP-18, or anycombination thereof; CTGF-1, CTGF-2, CGTF-3, CTGF-4, or any combinationthereof; VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, or any combinationthereof; GDF-1, GDF-2, GDF-3, GDF-7, GDF-10, GDF-11, GDF-15, or anycombination thereof; CDMP-1, CDMP-2, LMP-1, LMP-2, LMP-3, and/or anycombination thereof; Osteoprotegerin; TGF-beta-1, TGF-beta-2,TGF-beta-3, or any combination thereof; or any combination of one ormore members of these groups.

In some embodiments, the ligand is BMP-2, BMP-7 and/or GDF-5 and may beused at 1-2 mg/cc of matrix. The concentrations of ligand can be variedbased on the desired length or degree of osteogenic effects desired.However, the optimum ratio of ligand to receptor should be from about0.5 to about 1.5 or from about 0.7 to about 1.0 so that the ligand workswith optimum efficacy.

Similarly, one of skill in the art will understand that the duration ofsustained release of the ligand can be modified by the manipulation ofthe compositions of the matrix, such as for example, microencapsulationof the ligand within polymers. The sustained release matrix cantherefore be designed to provide customized time release of ligand thatstimulate the natural healing process.

The ligand may contain inactive materials such as buffering agents andpH adjusting agents such as potassium bicarbonate, potassium carbonate,potassium hydroxide, sodium acetate, sodium borate, sodium bicarbonate,sodium carbonate, sodium hydroxide or sodium phosphate;degradation/release modifiers; drug release adjusting agents;emulsifiers; preservatives such as benzalkonium chloride, chlorobutanol,phenylmercuric acetate and phenylmercuric nitrate, sodium bisulfate,sodium bisulfite, sodium thiosulfate, thimerosal, methylparaben,polyvinyl alcohol and phenylethyl alcohol; solubility adjusting agents;stabilizers; and/or cohesion modifiers. In some embodiments, the ligandmay comprise sterile and/or preservative free material.

These above inactive ingredients may have multi-functional purposesincluding the carrying, stabilizing and controlling the release of theligand and/or other therapeutic agent(s). The sustained release process,for example, may be by a solution-diffusion mechanism or it may begoverned by an erosion-sustained process.

In some embodiments, a pharmaceutically acceptable formulationcomprising a ligand is provided, wherein the formulation is afreeze-dried or lyophilized formulation, alone or in combination withthe matrix. Typically, in the freeze-dried or lyophilized formulation aneffective amount of a ligand is provided. Lyophilized formulations canbe reconstituted into solutions, suspensions, emulsions, or any othersuitable form for administration or use. The lyophilized formulation maycomprise the liquid used to reconstitute the ligand. Lyophilizedformulations are typically first prepared as liquids, then frozen andlyophilized. The total liquid volume before lyophilization can be less,equal to, or more than the final reconstituted volume of the lyophilizedformulation. The lyophilization process is well known to those ofordinary skill in the art, and typically includes sublimation of waterfrom a frozen formulation under controlled conditions.

Lyophilized formulations can be stored at a wide range of temperatures.Lyophilized formulations may be stored at or below 30° C., for example,refrigerated at 4° C., or at room temperature (e.g., approximately 25°C.).

Lyophilized formulations of the ligand are typically reconstituted foruse by addition of an aqueous solution to dissolve the lyophilizedformulation. A wide variety of aqueous solutions can be used toreconstitute a lyophilized formulation. In some embodiments, lyophilizedformulations can be reconstituted with a solution containing water(e.g., USP WFI, or water for injection) or bacteriostatic water (e.g.,USP WFI with 0.9% benzyl alcohol). However, solutions comprising buffersand/or excipients and/or one or more pharmaceutically acceptable carriescan also be used. In some embodiments, the solutions do not contain anypreservatives (e.g., are preservative free).

The matrix can also include a protease inhibitor to minimize degradationof the ligand and/or receptor. Protease inhibitors include saquinavir,indinavir, ritonavir, nelfinavir, amprenavir, lopinavir, atazanavir,fosamprenavir, tipranavir, darunavir, metalloprotease, MMP-1(collagenase-1), MMP-9, MMP-7 (matrilysin), MMP-8 (collagenase-2),MMP-13 (collagenase-3), MMP-18 (collagenase-4), MMP-2 (gelatinase a),MMP-9 (gelatinase b), MMP-3 (stromelysin-1), MMP-10 (stromelysin-2),MMP-11 (stromelysin-3), MMP-7 (matrilysin), MMP-26 (matrilysin), MMP-12(metalloelastase), MMP-14 (MT1-MMP), MMP-15 (MT2-MMP), MMP-16 (MT3-MMP),MMP-17 (MT4-MMP), MMP-24 (MT5-MMP) transmembrane, MMP-25 (MT6-MMP), gplanchor, MMP-19, MMP-20 (enamelysin), MMP-x, MMP-23, MMP-27, MMP-28(epilysin), serine protease inhibitors, such as for example, batimastat,aprotinin, or the like.

In various embodiments, the protease inhibitor can be in the matrix inan amount from approximately 0.0005 to approximately 100 μg/day toreduce degradation of the ligand and/or receptor. Other concentrationsof the protease inhibitor include from approximately 0.0005 toapproximately 50 μg/day; approximately 0.0005 to approximately 25μg/day; approximately 0.0005 to approximately 10 μg/day; approximately0.0005 to approximately 5 μg/day; approximately 0.0005 to approximately1 μg/day; approximately 0.0005 to approximately 0.75 μg/clay;approximately 0.0005 to approximately 0.5 μg/day; approximately 0.0005to approximately 0.25 μg/day; approximately 0.0005 to approximately 0.1μg/day; approximately 0.0005 to approximately 0.075 μg/day;approximately 0.0005 to approximately 0.05 μg/clay; approximately 0.001to approximately 0.025 μg/clay; approximately 0.001 to approximately0.01 μg/day; approximately 0.001 to approximately 0.0075 μg/day;approximately 0.001 to approximately 0.005 μg/day; approximately 0.001to approximately 0.025 μg/day; and approximately 0.002 μg/day. Inanother embodiment, the dosage of fluocinolone is from approximately0.001 to approximately 15 μg/day. In another embodiment, the proteaseinhibitor can be in a concentration in the matrix in an amount of fromapproximately 0.001 to approximately 10 μg/day or approximately 0.001 toapproximately 5 μg/day or from approximately 0.001 to 2.5 μg/day orbetween 40 and 600 μg/day or between 200 and 400 μg/day.

In some embodiments, if the protease inhibitor is an inorganic enzymeinhibitor, such for example, calcium the wide range of concentrationcould be used for example, the protease inhibitor can be in the matrixin an amount of 0.1%, 0.25%, 0.5%, 1.0%, 2.0%, 2.5%, 3%, 4%, 5%, 10%,15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% by weight based on the totalweight of the matrix to reduce the ligand and/or receptor degradation.In some embodiments, the protease inhibitor can be organic (e.g.,aprotinin) and be in the matrix in an amount of from about 1 mM, 1.5 mM,2 mM, 3 mM, or 5 mM, to about 0.1% by weight to reduce degradation ofthe ligand and/or receptor.

In various embodiments, the protease inhibitor reduces degradation ofthe ligand by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more so that the ligandlasts from 7, 10, 14, 30, 45, 60, 90, 180 days or longer.

Ligand Receptor Interactions

Receptors can include, for example, cell surface receptors such as Gprotein coupled receptors, integrins, growth factor receptors (e.g.,BMPRs), cytokine receptors, or can be the binding partner for theligand. Receptors can include antibodies, other polypeptides or ligandsof the immune system. Such other polypeptides of the immune systeminclude, for example, T cell receptors (TCR), major histocompatibilitycomplex (MHC), CD4 receptor or CD8 receptor. Furthermore, cytoplasmicreceptors such as steroid hormone receptors and DNA binding polypeptidessuch as transcription factors and DNA replication factors are likewiseincluded.

A ligand generally interacts with a receptor through multiple molecularinteractions resulting from multiple contact points or through multipleinteractions of a chemical functional group that can be described, forexample, as three points. These three points can be, for example, threedistinct chemical groups that serve as contact points for the bindingpartner. Likewise, three different amino acids or three differentclusters of amino acids in a polypeptide ligand or receptor can serve ascontact points for the binding partner. In this case, binding betweenthe ligand and receptor may occur when all three points can bind.

A receptor can be any molecule that binds to a ligand. The receptors canbe, for example, cell surface receptors that transmit intracellularsignals upon binding of a ligand. For example, the G protein coupledreceptors span the membrane seven times and couple signaling tointracellular heterotrimeric G proteins. G protein coupled receptorsparticipate in a wide range of physiological functions, includinghormonal signaling, vision, taste and olfaction. Moreover, thesereceptors encompass a large family of receptors, including receptors foracetylcholine, adenosine and adenine nucleotides, beta-adrenergicligands such as epinephrine, angiotensin, bombesin, bradykinin,cannabinoids, chemokines, dopamine, endothelin, histamine,melanocortins, melanotonin, neuropeptide Y, neurotensin, opioidpeptides, platelet activating factor, prostanoids, serotonin,somatostatin, tachykinin, thrombin and vasopressin, among others.

Other cell surface receptors have intrinsic tyrosine kinase activity andinclude growth factor or hormone receptors for ligands such asplatelet-derived growth factor, epidermal growth factor, insulin,insulin-like growth factor, hepatocyte growth factor, and other growthfactors and hormones. In addition, cell surface receptors that couple tointracellular tyrosine kinases include cytokine receptors such as thosefor the interleukins and interferons.

For example, when dealing with a ligand, such as bone morphogenicprotein (BMP), BMP interacts with specific receptors on the cellsurface, referred to as bone morphogenetic protein receptors (BMPRs). Aspersons of ordinary skill are aware the BMP spur the patient's body tobegin the formation of new bone and/or cartilage. The BMP acts much likea catalyst, encouraging the necessary cells (including, but not limitedto, mesenchymal stem cells, osteoblasts, and osteoclasts) to morerapidly migrate into the matrix, which is eventually resorbed via acell-mediated process and newly formed bone is deposited at or near thebone defect. In this manner severe fractures may be healed, andvertebrae successfully fused. Signal transduction through BMPRs resultsin mobilization of members of the SMAD family of proteins. The signalingpathways involving BMPs, BMPRs and SMADS are also important in thedevelopment of the heart, and central nervous system tissue, as well asbone and cartilage.

Other types of receptors include integrins. For example, integrins arecell surface receptors involved in a variety of physiological processessuch as cell attachment, cell migration and cell proliferation.Integrins mediate both cell-cell and cell-extracellular matrix adhesionevents. Structurally, integrins comprise a heterodimeric polypeptideswhere a single alpha chain polypeptide noncovalently associates with asingle beta chain. In general, different binding specificities arederived from unique combinations of distinct alpha and beta chainpolypeptides. For example, vitronectin binding integrins contain thealpha integrin subunit and include alpha beta₃, alpha beta_(v) and alphabeta₅, all of which exhibit different ligand binding specificities.

Receptors also can function in the immune system. An antibody orimmunoglobulin is an immune system receptor which binds to a ligand. Thepolypeptide receptor can be the entire antibody or it can be anyfunctional fragment thereof which binds to the ligand. Functionalfragments such as Fab, F(ab)₂, Fv, single chain Fv (scFv) and the likeare included within the definition of the term antibody. The use ofthese terms in describing functional fragments of an antibody areintended to correspond to the definitions well known to those skilled inthe art. Such terms are described in, for example, Harlow and Lane,Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York(1989). As with the above terms used for describing antibodies andfunctional fragments thereof, the use of terms which reference otherantibody domains, functional fragments, regions, nucleotide and aminoacid sequences and polypeptides or peptides, is similarly intended tofall within the scope of the meaning of each term as it is known andused within the art. Such terms include, for example, “heavy chainpolypeptide” or “heavy chain”, “light chain polypeptide” or “lightchain”, “heavy chain variable region” (V_(H)) and “light chain variableregion” (V_(L)) as well as the term “complementarity determining region”(CDR).

In addition to antibodies, the receptors can be T cell receptors (TCR).T cell receptors contain two subunits, alpha and beta, which are similarto antibody variable region sequences in both structure and function. Inthis regard, both subunits contain variable region which encode CDRregions similar to those found in antibodies (Immunology, Third Ed.,Kuby, J. (ed.), New York, W.H. Freeman & Co. (1997)). The CDR containingvariable regions of TCRs bind to antigens presented on the cell surfaceof antigen-presenting cells and are capable of exhibiting bindingspecificities to essentially any particular antigen.

Other exemplary receptors of the immune system which exhibit known orinherent binding functions include major histocompatiblility complex(MHC), CD4 and CD8. MHC functions in mediating interactions betweenantigen-presenting cells and effector T cells. CD4 and CD8 receptorsfunction in binding interactions between effector T cells andantigen-presenting cells. CD4 and CD8 also exhibit similar CDR regionstructure as do antibodies and TCRs sequences.

The ligand binds to its receptor and causes either directly orindirectly the biological function. The ligand can bind the receptorwhile it is attached to or in the matrix or alternatively it can bereleased from the matrix and be free at, near or in the target tissuesite until it binds the receptor.

The ligand should be in or on or released from the matrix so that itimproves efficacy and maintains a stable microenvironment for cellgrowth. By maintaining a ligand to receptor ratio of from about 0.5 toabout 1.5 the stable microenvironment for cell growth can be achieved.

The matrix is designed to provide an optimum ligand to receptor ratio sothat optimum binding is achieved. Optimum binding refers to a preferredbinding characteristic of a ligand and receptor interaction. Optimumbinding can be ligand-receptor interactions of a desired affinity,avidity or specificity. For example, optimum binding can be interactionsthat are most effective in the biological system (e.g., mammal). Theoptimum binding characteristics will depend on the particularapplication of the binding molecule. For example, the binding can berelative affinity of a ligand for the receptor. In this case, a ligandwith the highest binding affinity to a receptor would have optimumbinding. Optimum binding also can be binding to the largest number ofreceptor variants or binding to greater than some threshold number ofreceptor variants.

The concentration of ligand determines activity of receptors. Excessligand is not utilized, and may actually decrease endogenous ligand andother activities in a feed-back inhibition mechanism. In someembodiments, the ratio of ligand to receptor for most optimum bindingand efficiency is at or below 1.5 or 1. For example, in someembodiments, picking an average of 45,000 to 50,000 receptors per cell,a steady state capture rate of ligand to receptor of about 0.8 appearsto be optimal.

In some embodiments, the receptors are cell surface receptors disposedin or on progenitor, bone and/or cartilage cells.

In some embodiments, the concentration of ligand such as BMPs, otherTGF-beta ligands, TGF-alpha ligands, or EGF ligands on or in the matrixcan provide the desired biological result such as differentiatingmesenchymal stem cells into osteoblasts. The matrix allows the ligand tobe available at from about 0.2 nanograms to about 20 nanograms/ml ofmatrix/hour. For 7 days of activity from the matrix which is sufficientto convert enough mesenchymal stem cells to osteoblasts to produce boneafter maturation, and with degradation T1/2 of 5 minutes, 0.2×20×168=672ng/ml to 13,440 ng of ligand/ml of the matrix. This is 0.7 μg/ml to 14μg of ligand per ml of matrix. The availability of ligand in or on thematrix is therefore in line with the ability of receptors to accept theligand, and minimize feed-back inhibition and cross-activation ofosteoclasts by ligand or the target cells. If the degradation rate islower, less ligand is needed.

In some embodiments, the ligand can be replenished as it is depletedfrom the matrix. For examples, as the ligand gets depleted from thematrix by (i) degradation of the matrix, (ii) release of the ligand fromthe matrix, and/or (iii) binding of the ligand to the receptor on, near,or in the matrix, the matrix can be replenished with new ligand byinjection or infusion of the ligand into or on the matrix. The injectioncan be via a needled or cannulated device or it can be via an infusionpump that delivers the ligand in or on the matrix. In some embodiments,the matrix can also include a protease inhibitor to minimize degradationof the ligand and/or receptor.

In some embodiments, the ligand remains in the matrix or on its surfaceover a period of at least 3 days to 12 months.

Method of Making Matrix

In some embodiments, the matrix may be made by injection molding,compression molding, blow molding, thermoforming, die pressing, slipcasting, electrochemical machining, laser cutting, water-jet machining,electrophoretic deposition, powder injection molding, sand casting,shell mold casting, lost tissue scaffold casting, plaster-mold casting,ceramic-mold casting, investment casting, vacuum casting, permanent-moldcasting, slush casting, pressure casting, die casting, centrifugalcasting, squeeze casting, rolling, forging, swaging, extrusion,shearing, spinning, powder metallurgy compaction or combinationsthereof.

One form of manufacturing the matrix involves casting the matrixmaterial in a mold. The matrix material can take on the shape of themold such as, crescent, quadrilateral, rectangular, cylindrical, plug,or any other shape. Additionally, the surface of the mold may be smoothor may include raised features or indentations to impart features to thematrix. Features from the mold can be imparted to the matrix as thematrix material in the mold is dried. In particular aspects, a roughenedor friction engaging surface can be formed on the superior surfaceand/or the inferior surface of the matrix body. In some embodiments,protuberances or raised portions can be imparted on the superior surfaceand/or the inferior surface from the mold. Such examples ofprotuberances or raised portions are ridges, serrations, pyramids, andteeth, or the like.

In some embodiments, in manufacturing the matrix, a mixture of thematrix material (e.g., collagen) is combined with the elongatedparticles and a liquid to wet the material and form a slurry. Anysuitable liquid can be used including, for example, aqueous preparationssuch as water, saline solution (e.g. physiological saline), sugarsolutions, protic organic solvents, or liquid polyhydroxy compounds suchas glycerol and glycerol esters, or mixtures thereof. The liquid may,for example, constitute about 5 to about 70 weight percent of the mixedcomposition prior to the molding operation. Certain liquids such aswater can be removed in part or essentially completely from the formedmatrix using conventional drying techniques such as air drying, heateddrying, lyophilization, or the like.

In one embodiment of manufacture, a collagen mixture can be combinedwith a elongated particles and a liquid, desirably with an aqueouspreparation, to form a slurry. Excess liquid can be removed from theslurry by any suitable means, including for example by applying theslurry to a liquid-permeable mold or form and draining away excessliquid.

Before, during or after molding, including in some instances theapplication of compressive force to the collagen containing material,the collagen material can be subjected to one or more additionaloperations such as heating, lyophilizing and/or crosslinking to make theporous collagen interior or exterior of the matrix the desired porosity.In this regard, crosslinking can be used to improve the strength of theformed matrix. Alternatively, one or more of the surface of the matrixcan be crosslinked to reduce the size of the pores of the porousinterior and thereby form the exterior of the matrix that is lesspermeable and/or less porous than the porous interior. Crosslinking canbe achieved, for example, by chemical reaction, the application ofenergy such as radiant energy (e.g. UV light or microwave energy),drying and/or heating and dye-mediated photo-oxidation; dehydrothermaltreatment; enzymatic treatment or others.

Chemical crosslinking agents will generally be preferred, includingthose that contain bifunctional or multifunctional reactive groups, andwhich react with matrix. Chemical crosslinking can be introduced byexposing the matrix material to a chemical crosslinking agent, either bycontacting it with a solution of the chemical crosslinking agent or byexposure to the vapors of the chemical crosslinking agent. Thiscontacting or exposure can occur before, during or after a moldingoperation. In any event, the resulting material can then be washed toremove substantially all remaining amounts of the chemical crosslinkerif needed or desired for the performance or acceptability of the finalimplantable matrix.

Suitable chemical crosslinking agents include mono- and dialdehydes,including glutaraldehyde and formaldehyde; polyepoxy compounds such asglycerol polyglycidyl ethers, polyethylene glycol diglycidyl ethers andother polyepoxy and diepoxy glycidyl ethers; tanning agents includingpolyvalent metallic oxides such as titanium dioxide, chromium dioxide,aluminum dioxide, zirconium salt, as well as organic tannins and otherphenolic oxides derived from plants; chemicals for esterification orcarboxyl groups followed by reaction with hydrazide to form activatedacyl azide functionalities in the collagen; dicyclohexyl carbodiimideand its derivatives as well as other heterobifunctional crosslinkingagents; hexamethylene diisocyante; and/or sugars, including glucose,will also crosslink the matrix material.

In some embodiments, the matrices are formed by mixing the elongatedparticles in with a polymer slurry such as collagen and pouring into ashaped mold. The composite mixture is freeze dried and possiblychemically crosslinked and cut to the final desired shape.

In some embodiments, an implantable matrix is provided that isconfigured to fit at or near a target tissue site, the matrixcomprising: collagen and a plurality of elongated mineral particlesembedded within the collagen, the elongated mineral particles beingentangled with each other and embedded in the collagen uniformly orrandomly so as to reduce compression of the matrix, wherein the matrixallows influx of at least progenitor, bone and/or cartilage cellstherein; and the matrix comprises bone morphogenic protein.

In some embodiments, the matrix may comprise sterile and/or preservativefree material. The matrix can be implanted by hand or machine inprocedures such as for example, laparoscopic, arthroscopic,neuroendoscopic, endoscopic, rectoscopic procedures or the like.

The matrix of the present application may be used to repair bone and/orcartilage at a target tissue site, e.g., one resulting from injury,defect brought about during the course of surgery, infection, malignancyor developmental malformation. The matrix can be utilized in a widevariety of orthopedic, periodontal, neurosurgical, oral andmaxillofacial surgical procedures such as the repair of simple and/orcompound fractures and/or non-unions; external and/or internalfixations; joint reconstructions such as arthrodesis; generalarthroplasty; cup arthroplasty of the hip; femoral and humeral headreplacement; femoral head surface replacement and/or total jointreplacement; repairs of the vertebral column including spinal fusion andinternal fixation; tumor surgery, e.g., deficit filling; discectomy;laminectomy; excision of spinal cord tumors; anterior cervical andthoracic operations; repairs of spinal injuries; scoliosis, lordosis andkyphosis treatments; intermaxillary fixation of fractures; mentoplasty;temporomandibular joint replacement; alveolar ridge augmentation andreconstruction; inlay implantable matrices; implant placement andrevision; sinus lifts; cosmetic procedures; etc. Specific bones whichcan be repaired or replaced with the implantable matrix herein includethe ethmoid, frontal, nasal, occipital, parietal, temporal, mandible,maxilla, zygomatic, cervical vertebra, thoracic vertebra, lumbarvertebra, sacrum, rib, sternum, clavicle, scapula, humerus, radius,ulna, carpal bones, metacarpal bones, phalanges, ilium, ischium, pubis,femur, tibia, fibula, patella, calcaneus, tarsal and/or metatarsalbones.

Application of the Ligand to the Matrix

Ligands can be bound to the matrix by, for example, ionic bonds,non-covalent bonds, hydrogen bonds and/or van der Waals forces. In someembodiments, the ligand is bound to the matrix by chemical and/orbiological means such as for example by lyophilization, antibody, shortpeptides that bind the ligand, GRAS chemistry (generally regarded assafe), such as by using glutamic acid, glycine, hyaluronic acid, filmforming chemicals (e.g., using biological or synthetic amines), or thelike.

For example, a sterile matrix can be treated aseptically using a sterilefilm forming chemistry after soaking the matrix with the ligand and adry matrix can be added to the sterile matrix surgically or by injectionto load the matrix with the ligand for the desired activity.Film-forming agents that can be applied to or used in the matrix includethose substances that leave a pliable, cohesive, and continuous coveringon the surface of the matrix or in it. In some embodiments, the filmfrom the film-forming agent has strong hydrophillic properties.Film-forming agents include polyvinylpyrrolidone (PVP), acrylates (e.g.,polyacrylic acid), pullulan, polyvinyl alcohol, acrylamides, orcopolymers thereof or mixtures thereof.

In some embodiments, the film-forming agent can be cellulose and itsderivatives, chitosan, collagen, starch, modified starch, and variousnatural gum polymers and their derivatives, polymers and copolymers ofmethacrylic acid, amphiphilic copolymers such as polyethylene glycol30-dipolyhydroxystearate, various silicone polymers and copolymers, andpolyurethane or its derivatives. Film-forming agents include coatings onor in the matrix and can be applied by spraying, brushing, or variousindustrial processes, which undergo film formation. In mostfilm-formation processes, a liquid coating of relatively low viscosityis applied to a solid substrate and is cured to a solid,high-molecular-weight, polymer-based adherent film possessing theproperties desired by the user. For most common applications, this filmhas a thickness ranging from 0.5 to 500 micrometers (0.0005 to 0.5millimeters, or 0.00002 to 0.02 inches). The film-forming agent can alsobe the agent used to make the matrix as discussed above in the matrixsection. The film forming agent can be in the matrix in an amount offrom about 0.1%, 0.25%, 0.5%, 1.0%, 2.0%, 2.5%, 3%, 4%, or 5% wt % basedon the total weight of the matrix.

In some embodiments, a therapeutic agent (including one or more ligands)may be disposed on or in the interior of the matrix by hand,electrospraying, ionization spraying or impregnating, vibratorydispersion (including sonication), nozzle spraying,compressed-air-assisted spraying, injecting, brushing, soaking,lyophilization, and/or pouring.

Application of the ligand to the matrix may occur at the time of surgeryor by the manufacturer or in any other suitable manner. For example, theligand may be further reconstituted using a syringe and the syringe canbe placed into the interior of the matrix via insertion of a needle orcannula (piercing the matrix) and placing it into the interior of thematrix and injecting the ligand so it is evenly distributed throughoutthe porous interior.

In some embodiments, the ligand may be applied to the matrix (i.e.,collagen) prior to combining the materials and forming it into the finalmatrix shape. Indeed, the ligand can be blended into the natural orsynthetic polymer (i.e., POE) and poured into molds of the final shapeof the matrix. Alternatively, the ligand, such as a bone morphogeneticprotein in a suitable liquid carrier, may be applied onto and/or intothe porous loaded matrix after forming it into the final shape bysoaking, dripping, injecting, spraying, etc.

In some embodiments, the interior of the matrix is loaded with theligand BMP that functions as an osteoinductive factor. Indeed, thepreferred osteoinductive factors are the recombinant human bonemorphogenetic proteins (rhBMPs) because they are available in unlimitedsupply and do not transmit infectious diseases. In some embodiments, thebone morphogenetic protein is a rhBMP-2, rhBMP-4, rhBMP-7, orheterodimers thereof.

Recombinant BMP-2 can be used at a concentration of about 0.4 mg/ml toabout 10.0 mg/ml, preferably near 1.5 mg/ml. However, any bonemorphogenetic protein is contemplated including bone morphogeneticproteins designated as BMP-1 through BMP-18. BMPs are available fromWyeth, Cambridge, Mass. and the BMPs and genes encoding them may also beprepared by one skilled in the art as described in U.S. Pat. No.5,187,076 to Wozney et al.; U.S. Pat. No. 5,366,875 to Wozney et al.;U.S. Pat. No. 4,877,864 to Wang et al.; U.S. Pat. No. 5,108,922 to Wanget al.; U.S. Pat. No. 5,116,738 to Wang et al.; U.S. Pat. No. 5,013,649to Wang et al.; U.S. Pat. No. 5,106,748 to Wozney et al.; and PCT PatentNos. WO93/00432 to Wozney et al.; WO94/26893 to Celeste et al.; andWO94/26892 to Celeste et al. All osteoinductive factors are contemplatedwhether obtained as above or isolated from bone. Methods for isolatingbone morphogenetic protein from bone are described, for example, in U.S.Pat. No. 4,294,753 to Urist and Urist et al., 81 PNAS 371, 1984.

In some embodiments, the lyophilized ligand (e.g., BMP) can be disposedin a vial by the manufacturer and then the surgeon can mix the diluentwith the lyophilized growth factor. The matrix then can be parenterallyadministered to the target tissue site. The term “parenteral” as usedherein refers to modes of administration which bypass thegastrointestinal tract, and include for example, intramuscular,intraperitoneal, intrasternal, subcutaneous, intra-operatively,intrathecally, intradiscally, peridiscally, epidurally, perispinally,intraarticular or combinations thereof.

The amount of ligand, (e.g., bone morphogenic protein) may be sufficientto cause bone and/or cartilage growth. In some embodiments, the ligandis rhBMP-2 and is contained in one or more matrices in an amount of from1 to 2 mg per cubic centimeter of the biodegradable matrix. In someembodiments, the amount of rhBMP-2 morphogenic protein is from 2.0 to2.5 mg per cubic centimeter (cc) of the biodegradable matrix.

In some embodiments, the ligand is supplied in a liquid carrier (e.g.,an aqueous buffered solution). Exemplary aqueous buffered solutionsinclude, but are not limited to, TE, HEPES(2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid), MES(2-morpholinoethanesulfonic acid), sodium acetate buffer, sodium citratebuffer, sodium phosphate buffer, a Tris buffer (e.g., Tris-HCL),phosphate buffered saline (PBS), sodium phosphate, potassium phosphate,sodium chloride, potassium chloride, glycerol, calcium chloride or acombination thereof. In various embodiments, the buffer concentrationcan be from about 1 mM to 100 mM. In some embodiments, the BMP-2 isprovided in a vehicle (including a buffer) containing sucrose, glycine,L-glutamic acid, sodium chloride, and/or polysorbate 80.

In some embodiments, upon implantation of the matrix or components thatcontact the matrix (e.g., plugs that are separate from the matrix onimplantation), compression of the matrix is reduced or eliminated. Asdiscussed above, if unwanted compression occurs, this causes the bufferfrom the ligand to leak from the matrix, which causes higherconcentrations of the ligand (e.g., 2 mg to 2.5 mg of rhBMP-2 per cc ofmatrix) to remain on the matrix. This high concentration of ligand maylead to local transient bone resorption and excess osteoclast formationand bone breakdown. This may result in poor integration of the matrixwith surrounding host tissue and a failed repair. Thus, by employing acompression resistant matrix, unwanted leakage is reduced or avoided. Insome embodiments, localized release of the ligand may cause localirritation to the surrounding tissue. In some embodiments, the leakingof ligand from the matrix may reduce a stable microenvironment for newbone and/or cartilage growth. It also may cause the matrix to fail toretain its full efficacy over time to maximally promote growth at atarget site.

FIG. 1 illustrates an axial cross-sectional view of the implantablematrix being injected at a target tissue site and the optimum ligandconcentration being placed in the matrix. Shown is an axial view of avertebral cross section 1 that is undergoing a spinal fusion procedure.A surgeon prepares a hole 3 in the posterior position of the outerannulus fibrosis band 5. The dimensions of the hole 3 are suitable forinserting various types of intervertebral disc implantable matrices 7.For any of these matrices 7 disclosed, or other such devices 7, theinitial step is forming a hole 3 in the outer bands 5 of the annulusfibrosis 9, and the final steps involve sealing the hole 3. The matrices7 may include ligands and, in particular, may include osteogenic growthagents such as BMP (bone morphogenetic protein) and rhBMP, particularlyBMP-2 and rhBMP-2; bFGF (basic fibroblast growth factor); IGF-1(insulin-like growth factor); PDGF (platelet-derived growth factor);TGF-beta-1 (transforming growth factor beta-1); VEGF (vascularendothelial growth factor); GDF (growth and differentiation factor); orcombinations thereof.

The matrix 7 may be a combination of natural bone graft 11 and aninterbody device 13, in which the bone graft material 11 or the matrixmay include therapeutically effective amounts of one or more ligands.The interbody device 13 may be located where the nucleous pulposusresided, and the bone graft 11 fills the remaining area 2 of theintervertebral disc space where normally there would be natural nucleouspulposus tissue. The matrix 7 may act as a natural intervertebral discand may provide a cushion and support for adjacent vertebrae. Althoughthe method of the present invention refers to the matrix 7 beingassociated with osteoinductive factor ligands to promote bone and tissuegrowth between adjacent vertebrae, the matrix 7 can also be associatedwith other ligands.

After the surgeon inserts the various components 11, 13 and the matrix 7into the cleaned disc space 2, the hole 3 may be closed by administeringa second matrix 15 at or near the disc 4. The second matrix has disposedon or in it a ligand, in this case, BMP that delivers the BMP at aligand to receptor ratio of from about 0.5 to 1.5. The matrix allows theligand to be available at from about 0.2 nanograms to about 20nanograms/ml of matrix/hour. For 7 days of activity, the matrix may beloaded with 0.7 ug/ml to 14 ug of ligand per ml of matrix. This will besufficient to convert enough mesenchymal stem cells to osteoblasts toproduce bone after maturation, and it assumes degradation half-life of 5minutes, or 0.2×20×168=672 ng/ml to 13,440 ng of ligand/ml of thematrix. This is 0.7 ug/ml to 14 ug of ligand per ml of matrix. Theavailability of ligand in or on the matrix is therefore in line with theability of receptors to accept the ligand, and minimize feed-backinhibition and cross-activation of osteoclasts by ligand or the targetcells. If the degradation rate is lower, less ligand is needed. Thematrix 15 is delivered to the area in or about the hole 3.

In some embodiments, the matrix 15 can be a non-porous tissue adherentgel, a bio-resorbable polymer, or the like, that cures in vivo. Thematrix 15 may be injected into the disc space located in the posteriorsection of the spine, for example the posterior region of the outerannulus fibrosis band 5 where the surgically prepared hole 3 is located,or, if the surgery is of the anterior sort, the outer anterior region ofthe annulus fibrosis band 5. The matrix 15 may be injected, for example,into the annular fibrosis 5 from the outer edge 9 of the annularfibrosis 5 to about 2 mm to about 5 mm into the annular fibrosis 5. Thismethod may be well suited for interbody fusion procedures about thelumbar region of the spine 17, but it is within the scope of theinvention whereby the matrix 15 can be used in other regions of thespine.

The BMP acts much like a catalyst, encouraging the necessary cells(including, but not limited to, mesenchymal stem cells, osteoblasts, andosteoclasts) to more rapidly migrate into the matrix, which iseventually resorbed via a cell-mediated process and newly formed bone isdeposited at or near the bone defect. As the ligand (e.g., BMP) isdepleted from the matrix as binding occurs with the receptor BMPRs, theligand can be replenished by injecting additional ligand into the matrixor by infusion to keep the ligand to receptor ratio of from about 0.5 toabout 1.5 for optimum bone repair. Although, the spinal site is shown,it will be understood that the matrix can be used in other areas of thebody and other target tissue types including bone, muscle, tendons,ligaments, blood vessels, etc.

FIG. 2 illustrates a longitudinal cross-sectional view of soft tissueand sub-chondral bone where the matrix is implanted into a bone defect.The matrix contains a ligand (e.g., growth factor) that interacts withthe receptors of the bone. A target site 20 for the matrix 30 isidentified. The target site 20 may be, for example, a defect insub-chondral bone 22. The defect 20 may be a hole created by a surgeon,trauma, disease, or otherwise. The defect is surrounded by native bone22.

The matrix 30 comprises a ligand at a ratio of from about 0.5 to about1.5, which is uniformly disposed in it. In some embodiments, the matrix30 completely fills the defect 20. The matrix 30 may include, forexample, therapeutically effective amounts of an osteoinductive growthfactor, such as rhBMP-2, embedded within a bioresorbable scaffolding,such as a mixture of collagen and calcium phosphate. As the scaffoldingis absorbed by the host bone 22, the growth factor is released at bindsits receptor 24. Concentrations of the growth factor may encourage bonegrowth 50 within the defect 20 and/or within the matrix, thusencouraging the autologous bone 22 to migrate into and fill the defect20. The ligand can be maintained at a ratio of from about 0.5 to about1.5 so that optimum bone formation can occur and avoid unwanted boneformation in unwanted areas such as within local soft tissue area 40.

Elongated Particles and Stem Cells

In some embodiments, the matrix comprises elongated particles. Theelongated particles offer better compression resistance because of theincreased interaction of the elongated particles than conventionalrounded or spherical particles. When elongated particles are embedded ina polymer matrix, the elongated particles are tethered along theirlength therefore resisting movement when compressed.

In some embodiments, the elongated particles comprise at least 10%, 15%,20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95% by weight of the matrix. In some embodiments, the particles arepredominantly elongated (powders, chips, fibers, cylinders, etc.). Insome embodiments, there are non-elongated particles (e.g., rounded orspherical) in the matrix. In some embodiments, the particles in thematrix consist solely of elongated particles or non-elongated particles.

In some embodiments, the particles (bone and non-bone particles) areelongated i.e., they possess relatively high median length to medianthickness ratios. In overall appearance, the elongate particles can bedescribed as filaments, fibers, threads, slender or narrow strips, etc.Thus, e.g., the elongate particles can possess a median length of fromabout 2 to about 20 mm, or a median width of from about 0.02 to about 5mm or the ratio of median length to median width is from about 10:1 toabout 1000:1. In some embodiments, the elongated particles have a medianlength of from about 1 to about 10 mm, the median width of the elongatedparticles is from about 0.04 to about 2 mm and the ratio of medianlength to median width is from about 20:1 to about 200:1.

If desired, the elongate particles can be graded into different sizes toreduce or eliminate any less desirable size(s) of particles that may bepresent.

In some embodiments, the porosity of the elongated particles comprisesfrom 0 to 50%, in some embodiments, the porosity of the elongatedparticles comprises 5% to 25%.

In some embodiments, the elongated particles are not entangled with eachother but contact each other and portions of each elongated particleoverlap in the matrix to provide compression resistance. In someembodiments, at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,or more of the elongated particles overlap each other in the matrix.

In some embodiments, the elongated particles are not aggregated (e.g.,they do not clump together in a mass) in the matrix.

In some embodiments, the elongated particles are randomly distributedthroughout the matrix. In other embodiments, the elongated particles areuniformly or evenly distributed throughout the matrix. In someembodiments, the elongated particles may be dispersed in the matrixusing a dispersing agent. In other embodiments, the elongated particlesmay be stirred in the polymer and the mechanical agitation willdistribute the particles in the matrix until the desired distribution isreached (e.g., random or uniform).

The elongate particles can be readily obtained by any one of severalmethods, e.g., by milling or shaving the surface of an entire bone orrelatively large section of bone. Employing a milling technique, one canobtain a mass of elongate particles containing at least about 20 weightpercent of particles coming within the aforesaid range of dimensions.

In some embodiments, the matrix may comprise a resorbable ceramic (e.g.,hydroxyapatite, tricalcium phosphate, bioglasses, calcium sulfate, etc.)tyrosine-derived polycarbonate poly (DTE-co-DT carbonate), in which thependant group via the tyrosine—an amino acid—is either an ethyl ester(DTE) or free carboxylate (DT) or combinations thereof.

In some embodiments, the matrix may be seeded with harvested bone cellsand/or bone tissue, such as for example, cortical bone, autogenous bone,allogenic bones and/or xenogenic bone. In some embodiments, the matrixmay be seeded with harvested cartilage cells and/or cartilage tissue(e.g., autogenous, allogenic, and/or xenogenic cartilage tissue). Forexample, before insertion into the target tissue site, the matrix can bewetted with the graft bone tissue/cells, usually with bone tissue/cellsaspirated from the patient, at a ratio of about 3:1, 2:1, 1:1, 1:3 or1:2 by volume. The bone tissue/cells are permitted to soak into thematrix provided, and the matrix may be kneaded by hand or machine,thereby obtaining a pliable consistency that may subsequently be packedinto the bone defect. In some embodiments, the matrix provides amalleable, non-water soluble carrier that permits accurate placement andretention at the implantation site. In some embodiments, the harvestedbone and/or cartilage cells can be mixed with the growth factor andseeded in the interior of the matrix.

In some embodiments, the elongated particles in the matrix comprise aresorbable ceramic, bone, synthetic degradable polymer, hyaluronic acid,chitosan or combinations thereof. In some embodiments, the elongatedparticles comprise cortical, cancellous, and/or corticocancellous,allogenic, xenogenic or transgenic bone tissue. The bone component canbe fully mineralized or partially or fully demineralized or combinationsthereof. The bone component can consist of fully mineralized orpartially or fully demineralized bone.

In some embodiments, the matrix may contain an inorganic material, suchas an inorganic ceramic and/or bone substitute material. Exemplaryinorganic materials or bone substitute materials include but are notlimited to aragonite, dahlite, calcite, amorphous calcium carbonate,vaterite, weddellite, whewellite, struvite, urate, ferrihydrate,francolite, monohydrocalcite, magnetite, goethite, dentin, calciumcarbonate, calcium sulfate, calcium phosphosilicate, sodium phosphate,calcium aluminate, calcium phosphate, hydroxyapatite, alpha-tricalciumphosphate, dicalcium phosphate, β-tricalcium phosphate, tetracalciumphosphate, amorphous calcium phosphate, octacalcium phosphate,BIOGLASS™, fluoroapatite, chlorapatite, magnesium-substituted tricalciumphosphate, carbonate hydroxyapatite, substituted forms of hydroxyapatite(e.g., hydroxyapatite derived from bone may be substituted with otherions such as fluoride, chloride, magnesium sodium, potassium, etc.), orcombinations or derivatives thereof.

In some embodiments, by including inorganic ceramics, such as forexample, calcium phosphate, in the matrix, this will facilitate theprevention of local bone resorption by providing slower release of thegrowth factor due to its increased binding potential and also act as alocal source of calcium and phosphate to the cells attempting to depositnew bone. The inorganic ceramic also provides compression resistance andload bearing characteristics to the matrix.

In some embodiments, the elongated particles in the matrix comprisetricalcium phosphate and hydroxyapatite in a ratio of about 80:20 toabout 90:10. In some embodiments, the elongated particles in the matrixcomprise tricalcium phosphate and hydroxyapatite in a ratio of about85:15.

In some embodiments, tissue will infiltrate the matrix to a degree ofabout at least 50 percent within about 1 month to about 6 months afterimplantation of the matrix. In some embodiments, about 75 percent of thematrix will be infiltrated by tissue within about 2-3 months afterimplantation of the matrix. In some embodiments, the matrix will besubstantially, e.g., about 90 percent or more, submerged in or envelopedby tissue within about 6 months after implantation of the matrix. Insome embodiments, the matrix will be completely submerged in orenveloped by tissue within about 9-12 months after implantation.

In some embodiments, the matrix has a thickness of from 1 mm to 15 mm,or from about 2 mm to about 10 mm, or 3 mm to about 5 mm Clearly,different bone defects (e.g., osteochondral defects) may requiredifferent matrices thicknesses.

In some embodiments, the matrix has a density of between about 1.6g/cm³, and about 0.05 g/cm³. In some embodiments, the matrix has adensity of between about 1.1 g/cm³, and about 0.07 g/cm³. For example,the density may be less than about 1 g/cm³, less than about 0.7 g/cm³,less than about 0.6 g/cm³, less than about 0.5 g/cm³, less than about0.4 g/cm³, less than about 0.3 g/cm³, less than about 0.2 g/cm³, or lessthan about 0.1 g/cm³.

The shape of the matrix may be tailored to the site at which it is to besituated. For example, it may be in the shape of a morsel, a plug, apin, a peg, a cylinder, a block, a wedge, a sheet, a strip, etc. Theterm “shape” refers to a determined or regular form or configuration incontrast to an indeterminate or vague form or configuration (as in thecase of a lump or other solid mass of no special form) and ischaracteristic of such materials as sheets, plates, disks, cores, tubes,wedges, cylinders, or the like. This includes forms ranging fromregular, geometric shapes to irregular, angled, or non-geometric shapes,or combinations of features having any of these characteristics.

In some embodiments, the diameter or diagonal of the matrix can rangefrom 1 mm to 50 mm. In some embodiments, the diameter or diagonal of thematrix can range from 1 mm to 30 mm, or 5 mm to 10 mm which is smallenough to fit through an endoscopic cannula, but large enough tominimize the number of matrices needed to fill a large the bone defect(e.g., osteochondral defect). In some embodiments, at the time ofsurgery, the matrix can be cut by the surgeon to the desired shape tofit the tissue or bone defect and possibly hydrated with a growth factorif necessary.

In some embodiments, there is a method for treating a target tissue sitebeneath the skin in a patient in need of such treatment, the methodcomprising administering an implantable matrix configured to fit at ornear a target tissue site, the matrix comprising: a biodegradablecollagen and a ligand comprising bone morphogenic protein bound to thematrix and configured to bind a receptor of progenitor, bone and/orcartilage cells and allow influx of the cells into the implantablematrix, wherein the ratio of ligand to receptor is from about 1.0 toabout 0.5.

The matrices can be seeded with stem cells from any convenient source.However, stem cells that have osteogenic potential or that can betreated (e.g., differentiated) to generate cells with osteogenicpotential are preferred. Sources of stem cells that can be used in themethods, devices and matrices described herein include bone marrow,adipose tissue, muscle tissue, ex vivo cultured autologous mesenchymalstem cells, allogeneic off-the-shelf mesenchymal stem cells, umbilicalcord blood, embryonic yolk sac, placenta, umbilical cord, periosteum,fetal and adolescent skin, and blood. In some embodiments, the stemcells are mesenchymal stem cells or a mixture of cells that includemesenchymal stem cells (e.g., bone marrow aspirate). The stem cells canbe autologous, allogeneic or from xenogeneic sources. The stem cells canbe embryonic or from post-natal or adult sources.

Bone marrow aspirate is one source of stem cells useful in the methods,devices and matrices described herein. While such bone marrow aspiratecan be autologous, allogeneic or from xenogeneic sources, in someembodiments the bone marrow aspirate is autologous.

Bone marrow aspirate contains a complex mixture of hematopoietic stemcells, red and white blood cells and their precursors, mesenchymal stemand progenitor cells, stromal cells and their precursors, and a group ofcells including fibroblasts, reticulocytes, adipocytes, and endothelialcells which form a connective tissue network called “stroma.” Cells fromthe stroma morphologically regulate the differentiation of hematopoieticcells through direct interaction via cell surface proteins and thesecretion of growth factors and are involved in the foundation andsupport of the bone structure. Studies indicate that bone marrowcontains “pre-stromal” cells which have the capacity to differentiateinto cartilage, bone, and other connective tissue cells. Beresford“Osteogenic Stem Cells and the Stromal System of Bone and Marrow”, Clin.Orthop., 240:270, 1989.

In some embodiments, the stem cells include mesenchymal stem cells.Mesenchymal stem cells can be identified by procedures available tothose of skill in the art. For example, mesenchymal stem cells can beidentified via colony forming unit assays (CFU-f) or via flow cytometryusing markers that are typically expressed by mesenchymal stem cells.Mesenchymal stem cells generally express such markers as CD271+, CD105+,CD73+, but exhibit a CD34- and CD45-phenotype.

When bone marrow cells are employed, these cells may be obtained fromiliac crest, femora, tibiae, spine, rib or other medullary spaces. Insome embodiments, the stem cells are from an autologous fluid (e.g.,bone marrow aspirate). Bone marrow aspirate is a good source ofmesenchymal stem cells.

The stem cells can, in some embodiments, be subjected to a separationprocess such as centrifugation, size filtration, immunomageticselection, etc., in order to either screen out “irrelevant” cells, andimprove the efficacy of the efficacy of the mesenchymal stem cells tofacititate bone formation in implant materials. While it may not benecessary to separate the cell types and/or purify the mesenchymal stemcells, it some embodiments it may be desirable.

Additional Therapeutic Agents

The ligand of the present application may be disposed on or in thematrix with other therapeutic agents that can function as ligands orreceptors. For example, the ligand may be disposed on or in the carrierby electrospraying, ionization spraying or impregnating, vibratorydispersion (including sonication), nozzle spraying, soaking,compressed-air-assisted spraying, brushing, infusion, and/or pouring.

Exemplary therapeutic agents include but are not limited to IL-1inhibitors, such Kineret® (anakinra), which is a recombinant,non-glycosylated form of the human interleukin-1 receptor antagonist(IL-1Ra), or AMG 108, which is a monoclonal antibody that blocks theaction of IL-1. Therapeutic agents also include excitatory amino acidssuch as glutamate and aspartate, antagonists or inhibitors of glutamatebinding to NMDA receptors, AMPA receptors, and/or kainate receptors.Interleukin-1 receptor antagonists, thalidomide (a TNF-α releaseinhibitor), thalidomide analogues (which reduce TNF-α production bymacrophages), quinapril (an inhibitor of angiotensin II, whichupregulates TNF-α), interferons such as IL-11 (which modulate TNF-αreceptor expression), and aurin-tricarboxylic acid (which inhibitsTNF-α), may also be useful as therapeutic agents for reducinginflammation. It is further contemplated that where desirable apegylated form of the above may be used. Examples of still othertherapeutic agents include NF kappa B inhibitors such as antioxidants,such as dithiocarbamate, and other compounds, such as, for example,sulfasalazine.

Examples of therapeutic agents suitable for use also include, but arenot limited to, an anti-inflammatory agent, analgesic agent, orosteoinductive growth factor or a combination thereof. Anti-inflammatoryagents include, but are not limited to, apazone, celecoxib, diclofenac,diflunisal, enolic acids (piroxicam, meloxicam), etodolac, fenamates(mefenamic acid, meclofenamic acid), gold, ibuprofen, indomethacin,ketoprofen, ketorolac, nabumetone, naproxen, nimesulide, salicylates,sulfasalazine [2-hydroxy-5-[-4-[C2-pyridinylamino)sulfonyl]azo]benzoicacid, sulindac, tepoxalin, and tolmetin; as well as antioxidants, suchas dithiocarbamate, steroids, such as cortisol, cortisone,hydrocortisone, fludrocortisone, prednisone, prednisolone,methylprednisolone, triamcinolone, betamethasone, dexamethasone,beclomethasone, fluticasone or a combination thereof.

Suitable analgesic agents include, but are not limited to,acetaminophen, bupivicaine, fluocinolone, lidocaine, opioid analgesicssuch as buprenorphine, butorphanol, dextromoramide, dezocine,dextropropoxyphene, diamorphine, fentanyl, alfentanil, sufentanil,hydrocodone, hydromorphone, ketobemidone, levomethadyl, mepiridine,methadone, morphine, nalbuphine, opium, oxycodone, papavereturn,pentazocine, pethidine, phenoperidine, piritramide, dextropropoxyphene,remifentanil, tilidine, tramadol, codeine, dihydrocodeine, meptazinol,dezocine, eptazocine, flupirtine, amitriptyline, carbamazepine,gabapentin, pregabalin, or a combination thereof.

In some embodiments, a statin may be used. Statins include, but is notlimited to, atorvastatin, simvastatin, pravastatin, cerivastatin,mevastatin (see U.S. Pat. No. 3,883,140, the entire disclosure is hereinincorporated by reference), velostatin (also called synvinolin; see U.S.Pat. Nos. 4,448,784 and 4,450,171 these entire disclosures are hereinincorporated by reference), fluvastatin, lovastatin, rosuvastatin andfluindostatin (Sandoz XU-62-320), dalvastain (EP Appln. Publn. No.738510 A2, the entire disclosure is herein incorporated by reference),eptastatin, pitavastatin, or pharmaceutically acceptable salts thereofor a combination thereof. In various embodiments, the statin maycomprise mixtures of (+)R and (−)-S enantiomers of the statin. Invarious embodiments, the statin may comprise a 1:1 racemic mixture ofthe statin.

Kits

The matrix, ligand and devices to administer the implantable matrixcomposition may be sterilizable. In various embodiments, one or morecomponents of the matrix, and/or medical device to administer it may besterilizable by radiation in a terminal sterilization step in the finalpackaging. Terminal sterilization of a product provides greaterassurance of sterility than from processes such as an aseptic process,which require individual product components to be sterilized separatelyand the final package assembled in a sterile environment.

Typically, in various embodiments, gamma radiation is used in theterminal sterilization step, which involves utilizing ionizing energyfrom gamma rays that penetrates deeply in the device. Gamma rays arehighly effective in killing microorganisms, they leave no residues norhave sufficient energy to impart radioactivity to the device. Gamma rayscan be employed when the device is in the package and gammasterilization does not require high pressures or vacuum conditions,thus, package seals and other components are not stressed. In addition,gamma radiation eliminates the need for permeable packaging materials.

In some embodiments, the implantable matrix may be packaged in amoisture resistant package and then terminally sterilized by gammairradiation. In use the surgeon removes the one or all components fromthe sterile package for use.

In various embodiments, electron beam (e-beam) radiation may be used tosterilize one or more components of the matrix. E-beam radiationcomprises a form of ionizing energy, which is generally characterized bylow penetration and high-dose rates. E-beam irradiation is similar togamma processing in that it alters various chemical and molecular bondson contact, including the reproductive cells of microorganisms. Beamsproduced for e-beam sterilization are concentrated, highly-chargedstreams of electrons generated by the acceleration and conversion ofelectricity.

Other methods may also be used to sterilize the implantable matrixand/or one or more components of the matrix, including, but not limitedto, gas sterilization, such as, for example, with ethylene oxide orsteam sterilization.

In various embodiments, a kit is provided comprising the ligand, matrix,and/or diluents. The kit may include additional parts along with theimplantable matrix combined together to be used to implant the matrix(e.g., wipes, needles, syringes, etc.). The kit may include the matrixin a first compartment. The second compartment may include a vialholding the ligand, diluent and any other instruments needed for thelocalized drug delivery. A third compartment may include gloves, drapes,wound dressings and other procedural supplies for maintaining sterilityof the implanting process, as well as an instruction booklet, which mayinclude a chart that shows how to implant the matrix afterreconstituting the growth factor. A fourth compartment may includeadditional needles and/or sutures. Each tool may be separately packagedin a plastic pouch that is radiation sterilized. A fifth compartment mayinclude an agent for radiographic imaging. A cover of the kit mayinclude illustrations of the implanting procedure and a clear plasticcover may be placed over the compartments to maintain sterility.

It will be apparent to those skilled in the art that variousmodifications and variations can be made to various embodimentsdescribed herein without departing from the spirit or scope of theteachings herein. Thus, it is intended that various embodiments coverother modifications and variations of various embodiments within thescope of the present teachings.

1. An implantable matrix configured to fit at or near a target tissuesite, the matrix comprising: a biodegradable material and a ligand boundto the matrix and configured to bind a receptor and allow influx ofcells into the implantable matrix, wherein the ratio of ligand toreceptor is from about 0.5 to about 1.5.
 2. An implantable matrixaccording to claim 1, wherein the matrix allows influx of at leastprogenitor, bone and/or cartilage cells therein.
 3. An implantablematrix according to claim 1, wherein the ligand is a growth factor andremains in or on a surface of the implantable matrix over a period of 3days to 12 months.
 4. An implantable matrix according to claim 1,wherein the ligand remains in or on a surface of the implantable matrixat a concentration of 0.2-20 ng/ml/hour.
 5. An implantable matrixaccording to claim 1, wherein ligand is bone morphogenic protein and thereceptor is disposed in progenitor, bone and/or cartilage cells.
 6. Animplantable matrix according to claim 5, wherein the ligand is bonemorphogenic protein-2 and the biodegradable material comprises collagen.7. An implantable matrix according to claim 1, wherein the biodegradablematerial comprises one or more of poly(lactide-co-glycolide) (PLGA),polylactide (PLA), polyglycolide (PGA), D-lactide, D,L-lactide,L-lactide, D,L-lactide-co-ε-caprolactone,D,L-lactide-co-glycolide-co-ε-caprolactone, resorbable ceramic, bone,collagen, hyaluronic acid, glycine, glutamic acid or a combinationthereof.
 8. An implantable matrix according to claim 1, wherein thematrix further comprises an agent that reduces degradation of the ligandor a film-forming agent.
 9. An implantable matrix according to claim 1,wherein the agent that reduces degradation of the ligand comprises ametalloprotease or serine protease inhibitor.
 10. An implantable matrixaccording to claim 1, wherein the ligand remains in or on a surface ofthe implantable matrix at a concentration of 0.7 micrograms/ml to 14micrograms per ml of matrix.
 11. An implantable matrix according toclaim 7, wherein the resorbable ceramic comprises tricalcium phosphateand hydroxyapatite in a ratio of about 80:20 to about 90:10.
 12. Animplantable matrix according to claim 1, wherein the matrix is seededwith mesenchymal stem cells.
 13. A method for treating a target tissuesite beneath the skin in a patient in need of such treatment, the methodcomprising administering an implantable matrix configured to fit at ornear a target tissue site, the matrix comprising: a biodegradablecollagen and a ligand comprising bone morphogenic protein bound to thematrix and configured to bind a receptor of progenitor, bone and/orcartilage cells and allow influx of the cells into the implantablematrix, wherein the ratio of ligand to receptor is from about 0.5 toabout 1.5.
 14. A method according to claim 13, wherein the ligand is agrowth factor and remains in or on a surface of the implantable matrixover a period of 3 days to 12 months.
 15. A method according to claim13, wherein the ligand remains in or on a surface of the implantablematrix at a concentration of 0.2-20 ng/ml/hour.
 16. A method accordingto claim 13, wherein the bone morphogenic protein is bone morphogenicprotein-2.
 17. A method according to claim 13, wherein the bonemorphogenic protein to the receptor is maintained at a ratio of fromabout 1.0 to about 0.5.
 18. A method according to claim 13, wherein thematrix further comprises an agent that reduces degradation of theligand.
 19. A method of making an implantable matrix configured to fitat or near a target tissue site, the method comprising providing abiodegradable material and applying a ligand to bind the ligand to thematrix, the matrix configured to bind a receptor and allow influx ofcells into the implantable matrix, wherein the ligand is applied to thematrix in an amount where the ratio of ligand to receptor is from about0.5 to about 1.5.
 20. A method according to claim 19, further comprisingapplying an agent that reduces degradation of the ligand.